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Modification of internal configuration of a bubble column, airlift and stirred tank reactor (10-200 L) was made for root cultures of Bupleurum falcatum L. Agitation with an impeller covered with partition mesh was ineffective for a 10-L modified reactor, because it caused intensive foaming and subsequent overflow of the culture medium even at a low rotation speed of 50 rpm and a low aeration rate of 0.1 vvm (volume per volume of medium). In contrast, efficient aeration through a ceramic sparger placed at the bottom of a 20-L bubble column reactor yielded approximately 25 g/L of dry roots and 500 mg/L of saikosaponin-a and saikosaponin-d over 42 days. On a 200-L scale, however, the roots became flocculated under the upper perforated plate initially positioned near the middle of the reactor, forming a firm disk of roots and a large empty space between the disk and the medium. Thus, the roots had poor contact with the medium, which severely suppressed their growth. To avoid this flocculation, a bottom perforated plate and draft tube were installed as a partitioning device separating the culturing area (outside the draft tube) from the aeration area (inside the draft tube). The draft tube was made of a stainless steel mesh rather than a solid material, and the tube greatly increased the root yield in the 20-L reactor. This configuration was successfully applied at the 200-L scale, yielding 500-600 mg/L of saikosaponin-a and saikosaponin-d over 56 days.

Squamous cell carcinoma antigen 1 (SCCA1), which belongs to serine proteinase inhibitor (serpin) superfamily, inhibits papain-like cysteine proteinase. Recently, it has been reported that SCCA1 acts not only as a proteinase inhibitor but also as an inhibitor of UV-induced apoptosis via suppression of the activity of c-Jun NH(2)-terminal kinase (JNK1). The present study determined the crystal structure of SCCA1, suggesting that the reactive center loop (RCL) of SCCA1, a recognition site of proteinase, is very flexible and located away form the main-body of SCCA1. We show that the inhibitory effect of SCCA1 on the kinase activity of JNK1 is lost when the RCL was truncated. Furthermore, we found that a mutant protein created by replacing one amino acid in RCL maintain the suppressive activity to JNK1, whereas the inhibitory effect to proteinase is obviously decreased.

Chemical peeling with salicylic acid in polyethylene glycol vehicle (SA-PEG), which specifically acts on the stratum corneum, suppresses the development of skin tumors in UVB-irradiated hairless mice. To elucidate the mechanism through which chemical peeling with SA-PEG suppresses skin tumor development, the effects of chemical peeling on photodamaged keratinocytes and cornified envelopes (CEs) were evaluated in vivo. Among UVB-irradiated hairless mice, the structural atypia and expression of p53 protein in keratinocytes induced by UVB irradiation were intensely suppressed in the SA-PEG-treated mice 28 days after the start of weekly SA-PEG treatments when compared to that in the control UVB-irradiated mice. Incomplete expression of filaggrin and loricrin in keratinocytes from the control mice was also improved in keratinocytes from the SA-PEG-treated mice. In photo-exposed human facial skin, immature CEs were replaced with mature CEs 4 weeks after treatment with SA-PEG. Restoration of photodamaged stratum corneum by treatment with SA-PEG, which may affect remodeling of the structural environment of the keratinocytes, involved the normalization of keratinocyte differentiation and suppression of skin tumor development. These results suggest that the stratum corneum plays a protective role against carcinogenesis, and provide a novel strategy for the prevention of photo-induced skin tumors.

Background: Seasonal changes affect the condition of skin and may trigger various cutaneous disorders. Objective: To clarify the effects of the environmental humidity on the skin pathology, we studied the effects of the humidity on a water-holding function of the stratum corneum. Methods: We evaluated the skin surface conductance, amino acid in the stratum corneum, and immunoreactivity of filaggrin of the epidermis of hairless mice kept in different environmental humidity. Results: Skin surface conductance in the stratum corneum of hairless mice 3–7 days after transfer from a humid environment (>80% relative humidity) to a dry (

The epidermal permeability barrier of premature infants matures rapidly following birth. Previous studies suggest that air exposure could contribute to this acceleration, because: (i) development of a structurally and functionally mature barrier accelerates when fetal rat skin explants are incubated at an air-medium interface, and (ii) occlusion with a water-impermeable membrane prevents this acceleration. To investigate further the effects of air exposure on epidermal barrier ontogenesis, we compared the activities of several key enzymes of lipid metabolism and gene expression of protein markers of epidermal differentiation in fetal rat skin explants grown immersed versus air exposed. The rate-limiting enzymes of cholesterol (HMG CoA reductase) and ceramide (serine palmitoyl transferase) synthesis were not affected. In contrast, the normal developmental increases in activities of glucosylceramide synthase and cholesterol sulfotransferase, responsible for the synthesis of glucosylceramides and cholesterol sulfate, respectively, were accelerated further by air exposure. Additionally, two enzymes required for the final stages of barrier maturation and essential for normal stratum corneum function, beta-glucocerebrosidase, which converts glucosylceramide to ceramide, and steroid sulfatase, which desulfates cholesterol sulfate, also increased with air exposure. Furthermore, filaggrin and loricrin mRNA levels, and filaggrin, loricrin, and involucrin protein levels all increased with air exposure. Finally, occlusion with a water-impermeable membrane prevented both the air-exposure-induced increase in lipid enzyme activity, and the expression of loricrin, filaggrin, and involucrin. Thus, air exposure stimulates selected lipid metabolic enzymes and the gene expression of key structural proteins in fetal epidermis, providing a biochemical basis for air-induced acceleration of permeability barrier maturation in premature infants.

Lipids in the stratum corneum (SC) are organized into lamellar membrane unit structures that provide the permeability barrier. Cholesterol sulfate, a SC membrane lipid, is synthesized by cholesterol sulfotransferase (CSTase) in the lower epidermis and hydrolyzed to cholesterol by steroid sulfatase (SSase) in the SC. To determine whether these enzymes are induced during barrier ontogenesis, we examined their activity in epidermis of fetal rats before (gestational day 17), during (day 19), and after (day 21) barrier formation. CSTase activity increased ∼10-fold between day 17 and day 19 then declined between day 19 and day 21. In contrast, SSase activity reached its peak activity on day 21, increasing >5-fold. Fetal rat skin explants develop a SC and barrier over the same dine course in vitro as in utero . Likewise, CSTase and SSase activities during in vitro ontogenesis precisely mirrored those obtained in utero . Moreover, hormones that accelerate barrier ontogenesis ( e.g. glucocorticoids, thyroid hormone, and estrogen) accelerated the increase in CSTase and SSase activities during in vitro ontogenesis. mRNA levels of SSase increased in parallel with enzymatic activity, suggesting that these developmental changes are regulated at the genomic level. Finally, addition of exogenous cholesterol sulfate to explants in vitro did not accelerate either SC development or barrier formation. These studies suggest that induction of the cholesterol sulfate cycle enzymes during SC ontogenesis is a component of the fetal epidermal differentiation program and that the synthetic and degradative enzymes of this pathway are differentially regulated.