Shinji Inomata

Growth pattern and ginsenoside production of Agrobacterium-transformed Panax ginseng roots

Panax ginseng roots transformed by Agrobacterium rhizogenes grew rapidly in a hormone-free medium. The transformed roots showed biphasic growth: rapid during the first two weeks and slower thereafter. Sucrose in the medium was almost all converted to glucose and fructose during the first two weeks, and the root growth slowed down after the depletion of sucrose in the medium. Periodic changes of the medium maintained the high growth rate, and the dry weight increased by 31 times in 32 days, which is the highest growth rate so far reported for cultured tissues of ginseng. The medium exchange also increased the ginsenoside content in the roots. Effective scale-up of the root culture was achieved in a turbine-blade type bioreactor.

Coenzyme Q10 protects against oxidative stress-induced cell death and enhances the synthesis of basement membrane components in dermal and epidermal cells

Coenzyme Q10 (CoQ10), which has both energizing and anti‐oxidative effects, is also reported to have antiaging action, e.g., reducing the area of facial wrinkles. However, the mechanism of its anti‐aging activity is not fully established. Here, we examined the effect of CoQ10 on human dermal and epidermal cells. CoQ10 promoted proliferation of fibroblasts but not keratinocytes. It also accelerated production of basement membrane components, i.e., laminin 332 and type IV and VII collagens, in keratinocytes and fibroblasts, respectively; however, it had no effect on type I collagen production in fibroblasts. CoQ10 also showed protective effects against cell death induced by several reactive oxygen species in keratinocytes, but only when its cellular absorption was enhanced by pretreatment of the cells with highly CoQ10‐loaded serum. These results suggest that protection of epidermis against oxidative stress and enhancement of production of epidermal basement membrane components may be involved in the antiaging properties of CoQ10 in skin.

High-level production of arbutin from hydroquinone in suspension cultures of Catharanthus roseus plant cells

SummaryPlant cell suspensions of Catharanthus roseus efficiently converted exogenously supplied hydroquinone (HQ) into arbutin. Arbutin productivity of the cells was strongly influenced by the growth stage of the cultivated cells and by the manner of the addition of HQ. We have developed two methods: (i) cultivating suitable cells for producing arbutin at high density; (ii) efficiently adding toxic HQ to the cells. The yield of arbutin could be increased up to 9.2 g/l (45% of cell dry weight), which is the highest yield in the field of plant biotechnology. Repeated examinations and scaling up to a 20-l jar fermentor suggested that C. roseus cells stably produce arbutin in large amounts under the established conditions.

Glycylglycine decreases the size of conspicuous facial pores: Single-blinded half areas of face-applied study

Dear Editor, Enlarged, or dilated facial pores are one of the most frequently encountered skin problems in women of all ages. From our international survey, it has been revealed that more than half of women in their twenties and thirties complain of conspicuous pores. Because so little had been known about their anatomical structure, facial pores were investigated. The results indicated that pores were shown to form a cone-shaped hollow, which was rich in nucleated cells, an indication of parakeratosis. An investigation of the relationship between the degree of conspicuous pores and the condition of the skin indicated that women with abundant noticeably large pores showed high transepidermal water loss (TEWL) values, and high sebum content with significant amounts of unsaturated free fatty acids. A similar result of the relationship between the degree of visible pores and the amount of sebum has also been reported. Moreover, experimental application of oleic acid, an unsaturated fatty acid and the main component of human sebum, to the human skin promoted parakeratosis and increased TEWL values. Therefore, it was suggested that unsaturated fatty acids such as oleic acid may be responsible for the appearance of conspicuously enlarged pores. We then examined the effects of oleic acid on human keratinocytes to search for a substance that could inhibit such effects, and hopefully provide a way of improving the appearance of facial pores. When oleic acid was added to keratinocytes, it was found that the concentration of intracellular calcium ions increased. Various kinds of antagonists were applied before the addition of oleic acid, and N-methyl D-aspartate (NMDA)-type glutamate receptor antagonists such as MK801 were found to specifically inhibit the buildup of calcium ions. Concentrations of inflammatory cytokines such as interleukin (IL)-1α were increased by the addition of oleic acid, and MK801 inhibited the elevation of cytokine levels as such. These results suggested that oleic acid might cause parakeratosis around the pores with subsequent enlargement of the hollow, possibly via an NMDA receptor functioning in the corneocytes. We believed that the suppression of these effects would lead to the improvement of the appearance of facial pores. In addition to NMDA receptor antagonists, we screened for glycine-type receptor agonists, because glycine receptors are known to induce a chloride ion influx which should help cancel out the excess positive charge of the calcium ions in the cell. We detected that glycylglycine was one of the most effective compounds capable of suppressing increase of the TEWL value in human skin. Furthermore, glycylglycine was found to partially suppress the elevation of IL-1α levels induced by oleic acid. In this study we applied solutions containing 15% (w/w) ethanol with or without 1.6% (w/w) glycylglycine (Sigma-Aldrich, St Louis, MO, USA) to half areas of human cheeks of Japanese volunteers three times a day. We analyzed the areas corresponding to the hollow using replicas to establish whether or not glycylglycine can reduce the pore size and in turn improve the appearance of conspicuous pores. At first, 21 Japanese healthy men aged 20–50 years who had relatively conspicuous pores participated in April–May 2004, and application was performed for 1 month. Informed consent was obtained. Replicas with silicone rubber (Silflo, Flexico, Potters Bar, UK) were analyzed using a confocal microscope (HD100D; Lasertec, Yokohama, Japan). Pores were examined in areas of 3.3 × 3.3 mm of replicas by 3-D image analysis and the total areas of cross-section

Changes in the expression of epidermal differentiation markers at sites where cultured epithelial autografts were transplanted onto wounds from burn scar excision

This study investigated the recovery process during which grafted cultured epithelium formed normal epidermis. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6u2009weeks (stage 1), 5 samples after 6u2009weeks and within 6u2009months (stage 2), 6 samples after 6u2009months and within 18u2009months (stage 3) and 7 samples beyond 18u2009months (stage 4) after transplantation. These samples were stained for monoclonal antibodies against filaggrin, transglutaminase (TG), cytokeratin 6 and involucrin. Their expressions were examined in the epidermis. The expression patterns were classified using a six‐grade scale. The grades of filaggrin and TG were significantly higher at stage 3 and 4 compared with stage 1. There was a marginally significant increase in the grade of cytokeratin 6 at stage 3 and it was significantly higher at stage 4 compared with stage 1. These results showed that wound healing continued at a molecular level until the end of stage 3. The recovery of involucrin was delayed compared with that of other markers. TG and involucrin are thought to be regulated independently at the grafted sites.

Changes in fibrillin-1 expression, elastin expression and skin surface texture at sites of cultured epithelial autograft transplantation onto wounds from burn scar excision

This study investigated the recovery process during which grafted cultured epithelium generated skin elasticity and skin surface microarchitecture. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6 weeks (stage 1), 5 samples after 6 weeks and within 6 months (stage 2), 6 samples after 6 months and within 18 months (stage 3) and 7 samples beyond 18 months (stage 4) of transplantation. These samples were evaluated by taking replicas of skin surface, and histological changes of fibrillin‐1 and elastin. The expression patterns were classified using a grading scale. The grade of skin surface texture was significantly higher at stage 3 and marginally significantly higher at stage 4 compared with stage 1. The grade of fibrillin‐1 was marginally significantly higher at stage 3 and significantly higher at stage 4 compared with stage 1. The grade of elastin was marginally significantly higher at stage 4 compared with stage 1. These results showed that it is important for patients to have skin care and avoid external forces for at least 18 months after transplantation.