Aromatic oil from lavender as an atopic dermatitis suppressant.

In atopic dermatitis (AD), nerves are abnormally stretched near the surface of the skin, making it sensitive to itching. Expression of neurotrophic factor Artemin (ARTN) involved in such nerve stretching is induced by the xenobiotic response (XRE) to air pollutants and UV radiation products. Therefore, AD can be monitored by the XRE response. Previously, we established a human keratinocyte cell line stably expressing a NanoLuc reporter gene downstream of XRE. We found that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan metabolite and known inducer of the XRE, increased reporter and Artemin mRNA expression, indicating that FICZ-treated cells could be a model for AD. Lavender essential oil has been used in folk medicine to treat AD, but the scientific basis for its use is unclear. In the present study, we investigated the efficacy of lavender essential oil and its major components, linalyl acetate and linalool, to suppress AD and sensitize skin using the established AD model cell line, and keratinocyte and dendritic cell activation assays. Our results indicated that lavender essential oil from L. angustifolia and linalyl acetate exerted a strong AD inhibitory effect and almost no skin sensitization. Our model is useful in that it can circumvent the practice of using animal studies to evaluate AD medicines.

Bilirubin oxidase expression and activity enhancement from Myrothecium verrucaria in Aspergillus species.

We constructed a new Aspergillus expression vector (pSENSU2512nid) under the control of the enolase promoter with 12 tandem repeats of cis-acting elements (region III) and the heat shock protein 12 (Hsp12) 5' untranslated region (UTR). Bilirubin oxidase (EC: 1.3.3.5) from Myrothecium verrucaria, which catalyzes the oxidation of bilirubin to biliverdin, was overexpressed in Aspergillus oryzae and A. niger. The productivity was estimated to be approximately 1.2 g/L in the culture broth, which was approximately 6-fold higher than that of recombinant bilirubin oxidase (BOD) expressed in Pichia pastoris (Komagataella phaffii). BOD was purified using hydrophobic interaction chromatography, followed by ion exchange chromatography. The specific activity of the purified BOD against 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate was 57.6 U/mg and 66.4 U/mg for A. oryzae and A. niger, respectively. l-Ascorbic acid (4 mM) addition and storage under deoxygenated conditions for 3-7 d increased the specific activity of these Aspergillus-expressed BODs approximately 2.3-fold (154.1 U/mg). The BOD specific activity was enhanced by incubation at higher temperature (30-50 °C). Further characterization of the enzyme catalytic efficiency revealed that the Km value remained unchanged, whereas the kcat value improved 3-fold. In conclusion, this high-level of BOD expression meets the requirements for industrial-level production. Additionally, we identified an effective method to enhance the low specific activity during expression, making it advantageous for industrial applications.

Fucosyltransferase 8 (FUT8) and core fucose expression in oxidative stress response.

GlycoMaple is a new tool to predict glycan structures based on the expression levels of 950 genes encoding glycan biosynthesis-related enzymes and proteins using RNA-seq data. The antioxidant response, protecting cells from oxidative stress, has been focused on because its activation may relieve pathological conditions, such as neurodegenerative diseases. Genes involved in the antioxidant response are defined within the GO:0006979 category, including 441 human genes. Fifteen genes overlap between the glycan biosynthesis-related genes defined by GlycoMaple and the antioxidant response genes defined by GO:0006979, one of which is FUT8. 5-Hydroxy-4-phenyl-butenolide (5H4PB) extracted from Chinese aromatic vinegar induces the expression of a series of antioxidant response genes that protect cells from oxidative stress via activation of the nuclear factor erythroid 2-related factor 2-antioxidant response element pathway. Here, we show that FUT8 is upregulated in both our RNA-seq data set of 5H4PB-treated cells and publicly available RNA-seq data set of cells treated with another antioxidant, sulforaphane. Applying our RNA-seq data set to GlycoMaple led to a prediction of an increase in the core fucose of N-glycan that was confirmed by flow cytometry using a fucose-binding lectin. These results suggest that FUT8 and core fucose expression may increase upon the antioxidant response.

Evaluation of skin sensitization based on interleukin-2 promoter activation in Jurkat cells.

Skin sensitization is an allergic reaction caused by certain chemical substances, and is an important factor to be taken into consideration when evaluating the safety of numerous types of products. Although animal testing has long been used to evaluate skin sensitization, the recent trend to regulate such testing has led to the development and use of alternative methods. Skin sensitization reactions are summarized in the form of an adverse outcome pathway consisting of four key events (KE), including covalent binding to skin proteins (KE1), keratinocyte activation (KE2), and dendritic cell activation (KE3). Equivalent alternative methods have been developed for KE1 to KE3, but no valid alternative has yet been developed for the evaluation of KE4 and T-cell activation. Current alternative methods rely on data from KE1 to KE3 to predict the effect of chemicals on skin sensitization. The addition of KE4 data is expected to improve the accuracy and reproducibility of such predictions. The aim of this study was to establish an assay to evaluate KE4 T-cell activation to supplement data on skin sensitization related to KE4. To evaluate T-cell activation, the Jurkat T-cell line stably expressing luciferase downstream of the pro-inflammatory cytokine interleukin-2 promoter was used. After exposure to known skin sensitizing agents and control substances, luciferase activity measurements revealed that this assay was valid for evaluating skin sensitization. However, two skin sensitizers known to have immunosuppressive effects on T-cells reacted negatively in this assay. The results revealed that this assay simultaneously allows for monitoring of the skin sensitization and immuno-suppressiveness of chemical substances and supplements KE4 T-cell activation data, and may thus contribute to reducing the use of animal experiments.

Triclosan-lysozyme complex as novel antimicrobial macromolecule: a new potential of lysozyme as phenolic drug-targeting molecule.

A novel anti-infection strategy to alleviate antibiotic-resistance problem and non-specific toxicity associated with chemotherapy is explored in this study. It is based on utilizing a bacteriolytic enzyme (lysozyme) as a carrier to allow specific targeting of a potential phenolic antimicrobial drug (triclosan) to microbial cells. Lysozyme (LZ) was complexed, via electrostatic and hydrophobic condensation at alkaline pH, to various degrees with triclosan (TCS), a negatively charged phenolic antimicrobial that inhibits bacterial fatty acid synthesis. Fluorescence and absorbance spectra analysis revealed non-covalent association of TCS with the aromatic residues at the interior of LZ molecule. The conjugation greatly promoted the lytic activity of LZ as the degree of TCS derivatization increased. The complexation with LZ turned TCS into completely soluble in aqueous solution. TCS-LZ complexes showed significantly enhanced bactericidal activity against several strains of Gram-positive and Gram-negative bacteria compared to the activity of TCS or LZ alone when tested at the same molar basis. Strikingly, TCS-LZ complex, but not LZ or TCS alone, exhibited unique specificity to scavenge superoxide radicals, generated by the natural xanthine/xanthine oxidase coupling system, without affecting the catalytic function of oxidase. This finding is the first to describe that the membrane disrupting function of lysozyme can be utilized to specifically target antimicrobial drug(s) to pathogen cells and heralding a fascinating opportunity for the potential candidacy of TCS-LZ as novel antimicrobial strategy for human therapy.

Membrane permeation of giant unilamellar vesicles and corneal epithelial cells with lipophilic vitamin nanoemulsions.

Nanoemulsions of a lipophilic vitamin, retinol palmitate (vitamin A; VA), have a therapeutic effect on corneal damage. The nanoemulsion based on a triblock-type polymer surfactant with polyoxyethylene and polypropylene, EO100PO70EO100 (EOPO) showed superior efficacy, as compared with a nanoemulsion based on polyoxyethylene (60) hydrogenated castor oil (HCO). We studied the mechanism of VA nanoemulsions related to efficacy from the viewpoint of the interaction with plasma membrane-mimicking giant unilamellar vesicles (GUVs) and the plasma membrane permeation in corneal epithelial cells. When nanoemulsions and GUVs doped with fluorescent compounds were mixed each other, and observed by confocal laser microscopy, EOPO nanoemulsions induced endocytic morphological changes like strings and vesicles of the bilayer drawn inside a GUV by budding. Judging by isothermal titration calorimetry and ζ potential measurements, the EOPO nanoemulsions seemed to have stronger hydrophobic interactions with the lipid bilayer because of lower coverage of the core interface. Next, when the nanoemulsions prepared with a pyrene derivative of retinol (VApyr) were applied to corneal epithelial cells, the EOPO nanoemulsions greatly permeated the cells and gathered around the cell nucleus, as compared with HCO nanoemulsions. Furthermore, according to the three-dimensional images of the cell, it was found that the vesicles that absorbed nanoemulsions formed from the plasma membrane as real endocytosis, and were transported to the area around the nucleus. Consequently, it is likely that EOPO nanoemulsions entered the cell by membrane-mediated transport, delivering VA to the cell nucleus effectively and enhancing the effects of VA.

Fusion of single-photon emission computed tomography and computed tomography images of sentinel lymph nodes in extramammary Paget's disease of the scrotum.

We describe a new method for the fusion of single-photon emission computed tomography (SPECT) and multidetector computed tomography (MDCT) images using an acrylic resin marker containing 99 m-Tc phytate and viewing software (Fusion Viewer Version 1.0 for Windows). This method provided the fusion of SPECT and CT images in a short time and made it easy to identify the sentinel lymph nodes in the patient with extramammary Paget's disease of the scrotum.

A New Agonist for Peroxisome Proliferation-activated Receptor γ (PPARγ), Fraglide-1 from Zhenjiang Fragrant Vinegar: Screening and Characterization Based on Cell Culture Experiments.

Zhenjiang fragrant vinegar (Kozu) is a black rice vinegar that has been used as a traditional Chinese medicine and has various health benefits, including anti-obesity effects. In the present study, using luciferase reporter assays of PPARγ promoter activity, a novel ingredient from 8-year-old Kozu, 5-hydroxy-4-phenyl-butenolide, was isolated. The newly found agonist was named as "Fraglide-1". Moreover, in subsequent experiments, it was confirmed that fraglide-1 was an PPARγ agonist and it could increase expression level of the uncoupling protein (UCP)-1. Fraglide-1 was chemically synthesized and it was verified that expression of the PPARγ was increased in dose dependent manner. Although Kozu has been consumed globally as a functional food for thousands of years, the mechanisms behind its health effects have not been characterized. The active ingredient of Kozu was successfully found and the results unraveled a longtime mystery about Kozu for its beneficial health effect.

Chirality-Dependent Interaction of d- and l-Menthol with Biomembrane Models.

Chirality plays a vital role in biological membranes and has a significant effect depending on the type and arrangement of the isomer. Menthol has two typical chiral forms, d- and l-, which exhibit different behaviours. l-Menthol is known for its physiological effect on sensitivity (i.e. a cooling effect), whereas d-menthol causes skin irritation. Menthol molecules may affect not only the thermoreceptors on biomembranes, but also the membrane itself. Membrane heterogeneity (lipid rafts, phase separation) depends on lipid packing and acyl chain ordering. Our interest is to elaborate the chirality dependence of d- and l-menthol on membrane heterogeneity. We revealed physical differences between the two optical isomers of menthol on membrane heterogeneity by studying model membranes using nuclear magnetic resonance and microscopic observation.

Quantification of Fraglide-1, a New Functional Ingredient, in Vinegars.

Vinegar is a widely used condiment in the world, and is produced from ethanol by acetic acid fermentation. Different fruits, vegetables, cereals, and wines can be used as ingredients for vinegar production. It is known that vinegar has many nutrient components such as organic acids, polyphenols, and aromatic compounds. Because of these bioactive components, it has many health benefits. China has a long history of producing vinegar and has been using it for health products and as medicine. Chinese aromatic Zhenjiang vinegar (Kozu) is produced from sticky rice. It is famous for its special flavor and health benefits. 5-Hydroxy-4-phenyl-butenolide (Fraglide-1) is a functional compound discovered in Kozu and has anti-fungal and anti-obesity effects. In this study, the Fraglide-1 content of different kinds of vinegars and ingredients, including Kozu samples and ingredients, was investigated. Fraglide-1 analysis was carried out via LC-MS/MS in multiple reaction monitoring mode. It was found that all the Kozu samples, as well as brown rice vinegar (Kurosu) samples, and the Chinese sticky rice husk used for the production of Kozu, contained Fraglide-1. Kozu production requires a 6-month- to 8-year-long aging process for its special flavor and aroma. Because of this long aging process, Fraglide-1 is thought to move from the sticky rice husk to Kozu.

Antioxidant properties of 5-hydroxy-4-phenyl-butenolide via activation of Nrf2/ARE signaling pathway.

5-Hydroxy-4-phenyl-butenolide (5H4PB) is a bioactive compound with antifungal and anti-obesity properties. Although it has recently been shown that 5H4PB activates peroxisome proliferator-activated receptor-gamma (PPARγ), the effect of 5H4PB on intracellular signaling pathways has not been clarified. In this study, we found that 5H4PB activated the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway, which plays an important role in cellular defense against oxidative stress, and the subsequent upregulation of ARE-dependent cytoprotective genes, including the heme oxygenase-1, catalase, and superoxide dismutase genes, without exhibiting cytotoxicity. In addition, 5H4PB significantly attenuated intracellular ROS generation, glutathione oxidation, and DNA damage induced by hydrogen peroxide (H2O2) exposure in mouse fibroblast cells. Furthermore, we demonstrated that pretreatment with 5H4PB confers a significant cytoprotective effect against H2O2-induced cell death in mouse cultured fibroblasts and primary hepatocytes. Thus, our study demonstrated that 5H4PB enhanced cellular resistance to oxidative damage via activation of the Nrf2/ARE signaling pathway.

Raft-dependent endocytic movement and intracellular cluster formation during T cell activation triggered by concanavalin A.

Certain food ingredients can stimulate the human immune system. A lectin, concanavalin A (ConA), from Canavalia ensiformis (jack bean) is one of the most well-known food-derived immunostimulants and mediates activation of cell-mediated immunity through T cell proliferation. Generally, T cell activation is known to be triggered by the interaction between T cells and antigen-presenting cells (APCs) via a juxtacrine (contact-dependent) signaling pathway. The mechanism has been well characterized and is referred to as formation of the immunological synapse (IS). We were interested in the mechanism behind the T cell activation by food-derived ConA which might be different from that of T cell activation by APCs. The purpose of this study was to characterize T cell activation by ConA with regard to (i) movement of raft domain, (ii) endocytic vesicular transport, (iii) the cytoskeleton (actin and microtubules), and (iv) cholesterol composition. We found that raft-dependent endocytic movement was important for T cell activation by ConA and this movement was dependent on actin, microtubules, and cholesterol. The T cell signaling mechanism triggered by ConA can be defined as endocrine signaling which is distinct from the activation process triggered by interaction between T cells and APCs by juxtacrine signaling. Therefore, we hypothesized that T cell activation by ConA includes both two-dimensional superficial raft movement on the membrane surface along actin filaments and three-dimensional endocytic movement toward the inside of the cell along microtubules. These findings are important for developing new methods for immune stimulation and cancer therapy based on the function of ConA.

Higher radical scavenging activities of polyphenolic antioxidants can be ascribed to chemical reactions following their oxidation.

Radical scavenging activities of 34 natural antioxidants were investigated from an electrochemical viewpoint. While the correlation of the oxidation potentials with their DPPH radical scavenging activities (represented by EC(50)) was not high (the correlation coefficient, r=0.73), the number of electrons n required for oxidation of an antioxidant, being obtained by continuous flow-column electrolysis with a slower flow rate (0.05 ml min(-1)), did show a good correlation with EC(50) (1/EC(50)=1.67n+0.50 with r=0.94). The n values of most polyphenols were increased with a decrease in the flow rate, while those of nonpolyphenols were invariant. This suggests that a slower subsequent chemical reaction(s) should be involved in the oxidation of polyphenols, whose higher radical scavenging activities seem to be ascribed to the chemical reactions. In this study, we have proposed a possible mechanism for the oxidation of polyphenols, in which the oxidizable -OH moieties are reproduced through an oxidative dimerization (or more highly polymerization).

Skin analysis to determine causative agent in dermal exposure to petroleum products.

This study evaluates the usefulness of skin analysis to determine the causative agent in cases of dermal exposure. The study consists of an animal experiment and two human cases. The petroleum components detected at high concentrations in skin samples resembled the composition of those in the corresponding petroleum products. However, the petroleum components in blood were detected at low concentrations and were a different composition. Skin is considered to be an advantageous sample to estimate the petroleum product in clinical and forensic cases of dermal exposure.

A rapid and definitive diagnosis of kerosene dermatitis by an analysis of detached lesional epidermis using gas chromatography-mass spectrometry.

A 73-year-old woman, who suffered from erythema with bullae and pustules on her abdomen and anterior right thigh, visited our hospital without an awareness of the causative agents. The lesions appeared like first and second degree burns. The small amount of detached roof of bulla was sampled without skin biopsy before the ointment treatment. The sample was sonicated in an ultrasonic bath for 1 min in n-pentane, and then 1 mul of the extract was analyzed by gas chromatography-mass spectrometry (GC-MS). The causative agent was determined to be kerosene. An examination of blood samples collected at the first visit failed to detect kerosene, though traces of trimethylbenzene were detected. A GC-MS analysis of the small sample of lesional epidermis was very useful to identify kerosene as a causative agent.

Kinetic characteristics and toxic effects of benzalkonium chloride following intravascular and oral administration in rats.

Kinetic characteristics and toxic effects of benzalkonium chloride (BZK) following injection via jugular vein (JV), femoral artery (FA) and oral administration (PO) were experimentally investigated using rats. The BZK concentrations in blood and tissues (lung, liver and kidney) were determined by high-performance liquid chromatography with solid phase extraction. Toxic doses of 15 and 250 mg/kg of BZK were used for intravascular (JV and FA) and PO administration, respectively. The fatal effects appeared soon after the dose in JV-rats, while delayed in FA- or PO-rats. The blood BZK concentrations and the elimination half-lives were similar between JV- and FA-rats, while the distribution of BZK in tissues was slightly different. In PO administration, the rats that aspirated BZK into their lungs had some symptoms, while the rats that did not aspirate BZK appeared to be normal. The BZK concentrations in blood and tissues were significantly higher in the aspirated PO-rats. The toxic degree of BZK was correlated with the BZK concentration in orally dosed rats. Lung and kidney had higher BZK concentrations compared to blood or liver, and they could be the target organs of BZK.Keyword: Benzalkonium chloride

Dermal absorption of kerosene components in rats and the influence of its amount and area of exposure.

The influences of amount and area of dermal exposure to kerosene upon the levels of kerosene components in biological samples were examined in vivo and in vitro. Thirty-two rats were randomly divided into four groups and exposed to kerosene through the abdominal skin for 2h. The amounts (soaked in cotton) and area of kerosene exposed were 1 ml/4 cm(2) in Group I, 4 ml/4 cm(2) in Group II, 4 ml/16 cm(2) in Group III and 16 ml/64 cm(2) in Group IV. Before, then 5, 10, 20, 30, 45, 60, 90 and 120 min after exposure, 0.5 ml of blood was collected. Solid tissue samples, including the exposed skin area, were harvested at 120 min. Kerosene components were analyzed by gas chromatography/mass spectrometry. Trimethylbenzens (TMBs) that are easily absorbed kerosene components, appeared at 5-20 min. The time course changes in TMB levels in blood were significantly different between Groups I and II or Groups I and III, and almost identical between Groups II and III. Similar trends were observed in tissue samples at 120 min. High concentrations of aliphatic hydrocarbons (AHCs) were detected in the exposed skin and the AHC levels were dependent on the amount of kerosene exposed per unit area. These results suggest that (1) dermal absorption of kerosene occurs soon after dermal exposure started, (2) absorption of TMBs is influenced by the total amount of kerosene rather than area of exposure, and (3) AHCs remaining in the skin at significant levels are influenced by the amount of kerosene per unit area exposed.

The levels of kerosene components in biological samples after repeated dermal exposure to kerosene in rats.

The current study was experimentally investigated using rats whether or not kerosene components are accumulated from daily repeated dermal exposure. Rats received daily 1h-exposure to kerosene for 5 days (5K), daily 1h-exposure for 4 days and left for 1 day (4KL), a single 1h-exposure (1K), a single 1h-exposure and left for 1 day (1KL), or a single 1h-exposure, sacrificed and left dead for 1 day (1KLD). Kerosene components, trimethylbenzenes (TMBs) and aliphatic hydrocarbons (AHCs) in blood and tissues were determined by GC-MS. In blood, almost the same concentrations of TMBs were detected in the rats sacrificed immediately after exposure (5K, 1K and 1KLD), and only trace levels were detected in the rats sacrificed 1 day after exposure (4 and 1KL). Almost the same levels of AHCs in blood were detected among groups except for the rats sacrificed 1 day after a single exposure (1KL), in which AHCs were slightly lower. These results suggest that (1) AHCs tend to be accumulated from daily exposure, while TMBs do not, (2) the proportions of detected kerosene components in blood can be an indicator of whether the last exposure occurred just before death or not, (3) the kerosene levels last at least 1 day without blood circulation.

Atmospheric concentrations of sulfur dioxide, nitrogen oxides, ammonia, hydrogen chloride, nitric acid, formic and acetic acids in the south of Vietnam measured by the passive sampling method.

A passive sampling method was applied to measure the concentrations of air pollutants, such as sulfur dioxide, nitrogen oxides, ammonia, hydrogen chloride, and nitric, formic and acetic acids, in the ambient air at four test sites in the southern region of Vietnam. The monthly averages and the average concentrations of air pollutants during the period from July 2001 to September 2002 are reported here. The concentrations of air pollutants varied widely, depending on the test sites and the sampling periods. The average concentrations of sulfur dioxide in the air during the period from July 2001 to September 2002 at the four test sites were 3.3-16.9 ppb, and those of nitrogen dioxide were 5.4-12.8 ppb. The concentrations of nitrogen monoxide and hydrogen chloride were very low at all of the test sites. The observed concentrations of all of the above-mentioned pollutants were lower than those of the Vietnamese standards of air pollutants.