No. 3Vol. 42, No. 2, pp. 94–101, 2008Effect of Birch (Betula platyphylla Sukatchev var. japonica Hara) sap on cultured human epidermal keratinocyte differentiation

The beauty of ideal skin texture is closely associated with dermal moisture factors. The key factors of skin moisture are NMF (natural moisturizing factor) and skin normal barrier function. The former keeps dermal surface moisture, and the later protects from excess water loss. So we have searched for the ingredient that improves these factors. Birch sap has been widely used as an effective drink for anti-fatigue and anti-stress. However, the effect of birch sap on skin as a cosmetic agent has not been known entirely. In this study, we investigated the effects of birch ( Betula platyphylla Sukatchev var. japonica Hara) sap on human skin. Birch sap induced epidermal keratinocyte differentiation properties in vitro . We assessed two epidermal differentiation agents. Filaggrin is a precursor protein of NMF, and involucrin is one of the precursor proteins of the cornified cell envelope (CE), which is related to normal barrier function. We have evaluated the production of these proteins where birch sap was applied to human normal keratinocytes. Birch sap not only increased mRNA expression of filaggrin and involucrin, but also accelerated these proteins production. Otherwise, birch sap did not have any influence for IL-6 production, which is related to inflammatory and aberrant keratinocyte proliferation. The results of induced differentiation properties on birch sap-treated keratinocytes are very similar to the differentiation induced by calcium in vitro . This similarity suggested that birch sap has a differentiation inducible property on in vitro cultured keratinocytes. Our study suggested that birch sap is able to control both moisturizing- and barrier-related factor production. From these effects, birch sap provides appropriate epidermal functions and skin homeostasis, and revealed itself as a very useful ingredient in the cosmetic field.     

Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis

Many classes of nanoparticles have been synthesized and widely applied, however, there is a serious lack of information concerning their effects on human health and the environment. Considering that their use will increase, accurate and cost-effective measurement techniques for characterizing "nanotoxicity" are required. One major toxicological concern is that nanoparticles are easily taken up in the human body. In this study, we developed a method of evaluating the uptake potential of nanosized particles using flow cytometric light scatter. Suspended titanium dioxide (TiO2) particles (5, 23, or 5000 nm) were added to Chinese hamster ovary cells. Observation by confocal laser scanning microscopy showed that the TiO2 particles easily moved to the cytoplasm of the cultured mammalian cells, not to the nucleus. The intensity of the side-scattered light revealed that the particles were taken up in the cells dose-, time-, and size-dependently. In addition, surface-coating of TiO2 particles changed the uptake into the cells, which was accurately reflected in the intensity of the side-scattered light. The uptake of other nanoparticles such as silver (Ag) and iron oxide (Fe3O4) also could be detected. This method could be used for the initial screening of the uptake potential of nanoparticles as an index of "nanotoxicity".     

Quantitative bioimaging analysis of gonads in olvas-GFP/ST-II YI Medaka (transgenic Oryzias latipes) exposed to ethinylestradiol

This study investigates the adverse and persistent effects of ethinylestradiol (EE2) on mature gonads of transgenic olvas-GFPIST II-YI medaka (Oryzias latipes). The measurement of gonadal size calculating the GFP-fluorescent area was used as a technique that enabled monitoring gonads in living specimens by GFP fluorescence. First, mature medaka were exposed to EE2 (47.8-522 ng/L) for 4 weeks. The gonads showed a significant reduction of the GFP-fluorescent area and Gonadosomatic Index in males exposed to EE2 at >216 ng/L and females exposed at 522 ng/L. Histologically, males at all treatments exhibited testis-ova and additionally, high connective tissue prevalence at > or =216 ng/L. Next, mature male medaka were exposed to EE2 (43.7-473 ng/L) for 3 weeks and allowed to depurate for 6 weeks, to investigate persistent effects of EE2. Continuous gonad observation showed that GFP began to decline 3 weeks after initial exposure to > or =215 ng/L. After depuration, the gonad's fluorescent areas gradually recovered, with no statistical difference at the end of the depuration period; normal spermatogenesis was present in these individuals. Alterations in GFP fluorescence clearly indicate the condition of the gonad in transgenic medaka and this strain showed a facilitated screening fish model to detect the adverse effects on the gonad by estrogenic chemicals.     

Effect of chloride on the formation of 3-monochloro-1,2-propanediol fatty acid diesters and glycidol fatty acid esters in fish,meats and acylglycerols during heating

The effects of the presence of chloride on the formation of 3-monochloro-1,2-propanediol fatty acid esters (3-MCPDEs) and glycidol fatty acid esters (GEs) in saltwater fish, meats and acylglycerols (diacylglycerol and triacylglycerol) during heating were investigated in this study. Five saltwater fish species (salmon, saury, yellowtail, mackerel and Spanish mackerel) were grilled with a fish griller. 3-MCPDEs and GEs were detected in all of the grilled fish samples. The total amount of GEs was higher than 3-MCPDEs. Beef and pork patties with or without sodium chloride (1.5%) were cooked using gaseous fuel. The formation of 3-MCPDEs was significantly increased by the addition of sodium chloride to the meat patties, whereas the concentration of GEs in the cooked meat patties was not changed by the content of sodium chloride. Hexadecane solutions of diacylglycerol or triacylglycerol containing FeCl₃ were heated at 240°C. The formation of 3-MCPDEs was greatly increased by adding FeCl₃ to the solutions of triacylglycerol. The amounts of 3-MCPDEs decreased with the extension of the heating time. From these results, it is suggested that 3-MCPDEs and GEs are formed in saltwater fish and meats by cooking, and that the formation of 3-MCPDEs was affected by chloride in foodstuffs.     

New method for testing phototoxicity of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs), widespread environmental pollutants, were recently reported to show photomutagenesis. As contaminants in the environment are usually exposed to sunlight, a way to evaluate the phototoxic characteristics of pollutants is required. We have previously found that phosphorylation of histone H2AX (gamma-H2AX), which accompanied the induction of DNA double strand breaks (DSBs), was significantly induced by low concentrations of benzo[a]pyrene (10(-9)-10(-7) M) and UVA (0.6 J/cm2) in CHO-K1 cells. Higher concentrations have been required for the detection of DSBs. The aim of the present study is to investigate the applicability of gamma-H2AX in a new phototoxicity assay of PAHs. The human keratinocytes, HaCaT, were treated with four model PAHs (naphthalene, phenanthrene, pyrene, and benzo[a]pyrene, 10(-11)-10(-7) M) and/or UVA (5 J/cm2), and the induction of gamma-H2AX was assessed. Furthermore, DSBs were directly detected using a biased sinusoidal field gel electrophoresis, and the cell viability was examined as a general assay of phototoxicity. The induction of gamma-H2AX was detected in the presence of all the PAHs except naphthalene at concentrations of 10(-9)-10(-7) M, whereas neither DSBs nor cell death could be detected at those concentrations, and higher concentrations were required for the detection. Naphthalene showed no phototoxicity in any of the three different assays. These findings suggest that histone H2AX is a potential moleculartargetfor detecting the phototoxicity of PAHs more sensitively than the detection of cell viability and DSBs.     

Electromyography of Eating Apples: Influences of Cooking, Cutting, and Peeling

ABSTRACT: The mastication of 7 differently prepared apple samples (raw pieces peeled and unpeeled, sliced, grated, cooked pieces with and without peel, and half-cooked pieces, 10 g each) was evaluated using electromyography (EMG). Eleven subjects participated in the EMG recording of both sides of the masseter and temporal muscles while eating samples normally. Only the grated sample reduced the number of chewing strokes and muscle activity before swallowing. In contrast, the thin apple slices produced significantly shorter contraction duration and cycle time only during the 1st 5 chewing strokes. The EMG duration and cycle in subsequent chews, as well as the other parameters, did not significantly differ between slices and pieces. Cooked apples exhibited significantly lower EMG amplitude and muscle activity per chew than their raw counterparts; however, there was no evidence of reduced total muscle activity required for swallowing. Raw and cooked apples with peel yielded significantly greater EMG amplitude and longer duration than those without peel. These findings suggest that appropriate preparation is necessary for people with various mastication abilities: grated for very low ability, cooked for those with weak chewing force, and unpeeled for mastication training.     

Development of a genetic transformation system using new selectable markers for fission yeast Schizosaccharomyces pombe

We describe the development of a new transformation system, using multiple auxotrophic marker genes, for the fission yeast Schizosaccharomyces pombe. We developed three new auxotrophic marker genes (arg12(+), tyr1(+) and ade7(+)) and generated a new host strain, YF043, by Cre-loxP-mediated gene disruption. YF043 possessed six mutated biosynthetic genes (leu1-32, ura4-M190T, arg12::loxP, tyr1::loxP, ade7::loxP and his2::loxP). The combination of this host strain and the new selectable markers can be used for gene disruption using the same preexisting transformation systems. In addition, Sz. pombe vectors were constructed, containing selectable marker genes that complement the auxotrophies of YF043. These new vectors are available for gene disruption and heterologous protein expression in strain YF043. The new Sz. pombe host strain will be a useful tool for molecular genetic studies of Sz. pombe where multiple recombinant modifications or multiple mutations are needed.