ABSTRACT
Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.
Subject(s)
Cell Adhesion Molecules/metabolism , Keratinocytes/metabolism , Melanins/biosynthesis , Tyrosine/metabolism , Animals , Base Sequence , Cell Adhesion Molecules/genetics , Cell Line, Tumor , Humans , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , RNA, Small Interfering/genetics , alpha-MSH/metabolism , KalininABSTRACT
Actinobacillus succinogenes, which is known to produce large amounts of succinate during fermentation of hexoses, was able to grow on C4-dicarboxylates such as fumarate under aerobic and anaerobic conditions. Anaerobic growth on fumarate was stimulated by glycerol and the major product was succinate, indicating the involvement of fumarate respiration similar to succinate production from glucose. The aerobic growth on C4-dicarboxylates and the transport proteins involved were studied. Fumarate was oxidized to acetate. The genome of A. succinogenes encodes six proteins with similarity to secondary C4-dicarboxylate transporters, including transporters of the Dcu (C4-dicarboxylate uptake), DcuC (C4-dicarboxylate uptake C), DASS (divalent anionâ:âsodium symporter) and TDT (tellurite resistance dicarboxylate transporter) family. From the cloned genes, Asuc_0304 of the DASS family protein was able to restore aerobic growth on C4-dicarboxylates in a C4-dicarboxylate-transport-negative Escherichia coli strain. The strain regained succinate or fumarate uptake, which was dependent on the electrochemical proton potential and the presence of Na(+). The transport had an optimum pH ~7, indicating transport of the dianionic C4-dicarboxylates. Transport competition experiments suggested substrate specificity for fumarate and succinate. The transport characteristics for C4-dicarboxylate uptake by cells of aerobically grown A. succinogenes were similar to those of Asuc_0304 expressed in E. coli, suggesting that Asuc_0304 has an important role in aerobic fumarate uptake in A. succinogenes. Asuc_0304 has sequence similarity to bacterial Na(+)-dicarboxylate cotransporters and contains the carboxylate-binding signature. Asuc_0304 was named SdcA (sodium-coupled C4-dicarboxylate transporter from A. succinogenes).
Subject(s)
Actinobacillus/metabolism , Dicarboxylic Acid Transporters/metabolism , Gene Expression Regulation, Bacterial , Actinobacillus/genetics , Actinobacillus/growth & development , Aerobiosis , Amino Acid Sequence , Anaerobiosis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Transport , Carbon Radioisotopes/analysis , Dicarboxylic Acid Transporters/genetics , Dicarboxylic Acids/metabolism , Fumarates/metabolism , Glucose/metabolism , Models, Biological , Molecular Sequence Data , Phylogeny , Sequence Alignment , Sodium/metabolism , Succinates/metabolismABSTRACT
The improvement of bacterial tolerance to organic solvents is a main prerequisite for the microbial production of biofuels which are toxic to cells. For targeted genetic engineering of Escherichia coli to increase organic solvent tolerances (OSTs), we selected and investigated a total of 12 genes that participate in relevant mechanisms to tolerance. In a spot assay of 12 knockout mutants with n-hexane and cyclohexane, the genes fadR and marR were finally selected as the two key genes for engineering. Fatty acid degradation regulon (FadR) regulates the biosynthesis and degradation of fatty acids coordinately, and the multiple antibiotic resistance repressor (MarR) is the repressor of the global regulator MarA for multidrug resistance. In the competitive growth assay, the ΔmarR mutant became dominant when the pooled culture of 11 knockout mutants was cultivated successively in the presence of organic solvent. The increased OSTs in the ΔmarR and ΔfadR mutants were confirmed by a growth experiment and a viability test. The even more highly enhanced OSTs in the ΔfadR ΔmarR double mutant were shown compared with the two single mutants. Cellular fatty acid analysis showed that the high ratio of saturated fatty acids to unsaturated fatty acids plays a crucial role in OSTs. Furthermore, the intracellular accumulation of OST strains was significantly decreased compared with the wild-type strain.