ABSTRACT
Objective To construct the eukaryotic expression vector of pyruvate kinase M1 (PKM1) gene labeled with pXJ-40-myc and detect its biological activity in ocular B16 melanoma cells.Methods Ocular B16 melanoma ceils were randomly divided into experimental and control group,and the experimental group was transfected with pXJ-40-myc-PKM1 plasmid and the control group was transfected with pXJ-40-myc plasmid.Then PKM1 gene was amplified by PCR with human liver cDNA library as the template.The recombinant plasmid pXJ-40-myc-PKM1 was identified by bacteria PCR and double enzyme digestion,followed by transfection of pXJ40-myc-PKM1 and pXJ-40-myc plasmid into B16 melanoma cells,and finally,the expression of PKM1 protein was verified by the Western blot,while wound healing assay was used to detect the effects of PKM1 on the migration of ocular melanoma ceils.Results The length of PKM1 gene was 1800bp,which was consistent with the expected size.Compared with the control group,the result of bacteria PCR was positive.The length of double enzyme digestion was 4000 bp and 1800 bp respectively.Western blot results showed that recombinant plasmld pXJ-40-myc-PKM1 was successfully expressed in ocular B16 melanoma cells.Compared with the control group,wound healing assay showed that recombinant plasmid could inhibit the migration of ocular B16 melanoma cells.Conclusion The eukaryotic expression vector of pXJ-40-myc-PKM1 is successfully constructed,which can suppress the migration of ocular B16 melanoma cells.
ABSTRACT
Objective To construct the eukaryotic expression vector of human BRE1B labeled with pCMV-Tag-2B and detect its biological activity in melanoma cells preliminarily.Methods Ocular B16 melanoma cells were randomly divided into the experimental group,in which the cells were transfected with pCMV-Tag-2B-BRE1B and the control group,which was transfected with pCMV plasmid.The CDS coding region of human BRE1B gene was amplified by PCR using human mammary gland cDNA as a template for construction of the recombinant plasmid pCMV-Tag-2B-BRE1B.After transfected with pCMV-Tag-2B-BRE1B and pCMV plasmid in the experimental and control group,respectively,Western blot was applied to detect the expression of BRE1B protein,while cell counting kit-8 (CCK8) and colony assays were used to analyze the effects of recombinant plasmid pCMV-Tag-2B-BRE1B on the growth of B16 melanoma cells.Results The CDS coding sequence of human BRE1B gene was amplified by PCR successfully,which was equal to the expected size.Compared with the control group,the sequence from bacteria PCR was identified as positive,with the length of 4000 bp and 3050 bp by double enzyme digestion respectively.Moreover,the coding sequence of the human BRE1B gene was exactly the same as the inserted DNA sequence.Western blot results showed that the expression of recombinant plasmid pCMV-Tag-2B-BRE1B was successfully expressed in the experimental group,but there was no specific fragments in the control group.And cell counting kit-8 (CCK8) and colony assays showed that pCMV-Tag-2B-BRE1B recombinant plasmid could inhibit the growth of B16 melanoma cells.Conclusion The eukaryotic expression vector of pCMV-Tag-2B-BRE1B labeled with pCMV-Tag-2B is constructed successfully,and it has inhibitory effects on the growth of ocular B16 melanoma cells.
ABSTRACT
In continuing to search new materials for skin whitening agent from natural resources, we have focused on plants used by Dayak tribe (native people in East Kalimantan, Indonesia) for skin care treatment. The ethanol extract of the leaves of Sonneratia caseolaris showed anti-melanogenesis activity in melanin biosynthesis assays using B16 melanoma cells. By activity-guided fractionation, luteolin-7-O--glucoside (compound 1) was isolated as an active compound. In melanin formation inhibition assay, luteolin-7-O--glucoside showed the inhibitory activity in a dose-dependent manner and reach 44% of inhibition at 223.2 μM with 92% of cells viability. The compound also showed potent anti-oxidative stress ability with a significant intracellular H2O2-scavenging activity in HaCaT cells. These results showed a validation of traditional use for skin care treatment by Dayak tribe in East Kalimantan.
ABSTRACT
2-Hydroxytyrosol (2-HT), originally reported as a synthetic compound, was isolated for the first time as a fungal metabolite. 2-HT was found to inhibit mushroom tyrosinase with an IC50 value of 13.0 µmol/L. Furthermore, 2-HT dose-dependently inhibited tyrosinase activity (IC50, 32.5 µmol/L) in the cell-free extract of B16 melanoma cells and α-melanocyte stimulating hormone (α-MSH)-stimulated melanin formation in intact B16 melanoma cells.
ABSTRACT
Nuclear factor of activated T-cells (NFAT) proteins are, calcium-regulated transcription factors, key regulator of stimulation-dependent gene activation. In our microarray analysis for the genes expressed in human black and white hairs, NFAT2 was significantly upregulated in the white hair, compared to the black hair. The aim of this study was to investigate functional role of NFAT2 in melanogenesis. Western blot analysis was performed to investigate the expression of NFAT2 protein in B16 melanoma cells. Our data showed that NFAT2 expression was increased in the hypopigmented B16 cells, while tyrosinase and MITF expression was decreased. To investigate the potential role of NFAT2, the recombinant adenovirus expressing microRNA specific for NFAT2 was transduced into the cultured B16 melanoma cells. Consistently, inhibition of NFAT2 enhanced tyrosinase activity and melanin content. Moreover, cyclosporine A, which is known as a calcineurin inhibitor blocking NFAT activation, enhanced tyrosinase activity and melanin content. These data suggest that NFAT2 may play an important role in regulation of melanogenesis in melanocyte.
Subject(s)
Humans , Adenoviridae , Blotting, Western , Calcineurin , Cyclosporine , Down-Regulation , White People , Hair , Hydroquinones , Melanins , Melanocytes , Melanoma, Experimental , Microarray Analysis , MicroRNAs , Monophenol Monooxygenase , NFATC Transcription Factors , Proteins , T-Lymphocytes , Transcription Factors , Transcriptional ActivationABSTRACT
10000 u,SP2:6 000 u0.05),the melanogenesis and tyrosinase activity were inhibited remarkably(P
ABSTRACT
Objective:To study the effects of different molecular weight of collagen polypeptides from Apostichopus japonicus (A1:6 000U