Age-related changes in cyclic phosphatidic acid-induced hyaluronic acid synthesis in human fibroblasts.

Hyaluronic acid is a major component of the extracellular matrix, which is important for skin hydration. As aging brings skin dehydration, we aimed to clarify the mRNA expression of hyaluronic acid-related proteins in human skin fibroblasts from donors of various ages (range 0.7-69 years). Previously, we reported that cyclic phosphatidic acid (cPA), a unique phospholipid mediator, stimulated the expression of HAS2 and increased hyaluronic acid synthesis in human skin fibroblasts (donor age: 3 days). In this study, we measured the mRNA expression of hyaluronic acid-related proteins: hyaluronan synthase (HAS) 1-3, hyaluronidase-1, -2, and hyaluronic acid-binding protein (versican). In addition, we tested whether cPA could increase hyaluronic acid synthesis in skin fibroblasts derived from donors of various ages. The expression of HAS1, 3, hyaluronidase-1, and -2 did not change with aging. However, the mRNA expression of versican decreased with aging. Although it is thought that the amount of hyaluronic acid in the dermis decreases with aging, the mRNA expression of HAS2 was increased. But the amount of hyaluronic acid secreted by fibroblasts did not increase with aging. This suggests that the activity and/or protein expression of HAS2 decrease with aging. Furthermore, we observed that cPA caused the increase of hyaluronic acid synthesis at any age, and this effect was increased with aging. These results suggest that aging made the fibroblasts more sensitive to cPA treatment. Therefore, cPA represents a suitable candidate for the health maintenance and improvement of the skin by increasing the level of hyaluronic acid in the dermis.

Volatile Components of the Essential Oil of Artemisia montana and Their Sedative Effects.

The sedative effects of volatile components in the essential oil of Artemisia montana ("Yomogi") were investigated and measured using gas chromatography-mass spectrometry (GC-MS). Major components identified included 1,8-cineol, camphor, borneol, α-piperitone, and caryophyllene oxide. Among them, 1,8-cineol exhibited the highest flavor dilution (FD) value in an aroma extract dilution analysis (AEDA), followed by borneol, o-cymene, ß-thujone, and bornyl acetate. The sedative effects of yomogi oil aroma were evaluated by sensory testing, analysis of salivary α-amylase activity, and measurement of relative fluctuation of oxygenated hemoglobin concentration in the brain using near-infrared spectroscopy (NIRS). All results indicated the stress-reducing effects of the essential oil following nasal exposure, and according to the NIRS analysis, 1,8-cineol is likely responsible for the sedative effects of yomogi oil.

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis.

Humans are unable to synthesize l-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.

A bifunctional enzyme with L-fucokinase and GDP-L-fucose pyrophosphorylase activities salvages free L-fucose in Arabidopsis.

Monomeric sugars generated during the metabolism of polysaccharides, glycoproteins, and glycolipids are imported to the cytoplasm and converted to respective nucleotide sugars via monosaccharide 1-phosphates, to be reutilized as activated sugars. Because L-fucose (L-Fuc) is activated mainly in the form of GDP derivatives in seed plants, the salvage reactions for L-Fuc are expected to be independent from those for Glc, Gal, L-arabinose, and glucuronic acid, which are activated as UDP-sugars. For this study we have identified, in the genomic data base of Arabidopsis, the gene (designated AtFKGP) of a bifunctional enzyme with similarity to both L-fucokinase and GDP-L-Fuc pyrophosphorylase. Recombinant AtFKGP (rAt-FKGP) expressed in Escherichia coli showed both L-fucokinase and GDP-L-Fuc pyrophosphorylase activities, generating GDP-L-Fuc from L-Fuc, ATP, and GTP as the starting substrates. Point mutations in rAtFKGPs at either Gly(133) or Gly(830) caused loss of GDP-L-Fuc pyrophosphorylase and l-fucokinase activity, respectively. The apparent K(m) values of L-fucokinase activity of rAtFKGP for L-Fuc and ATP were 1.0 and 0.45 mm, respectively, and those of GDP-L-Fuc pyrophosphorylase activity for L-Fuc 1-phosphate and GTP were 0.052 and 0.17 mm, respectively. The expression of AtFKGP was detected in most cell types of Arabidopsis, indicating that salvage reactions for free L-Fuc catalyzed by AtFKGP occur ubiquitously in Arabidopsis. Loss-of-function mutants with tDNA insertion in AtFKGP exhibited higher accumulation of free L-Fuc in the soluble fraction than the wild-type plant. These results indicate that AtFKGP is a bifunctional enzyme with L-fucokinase and GDP-L-Fuc pyrophosphorylase activities, which salvages free L-Fuc in Arabidopsis.