• Molecules and Cells
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• v.46 no.9
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• pp.545-557
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• 2023

Sphingomyelinase (SMase) catalyzes ceramide production from sphingomyelin. Ceramides are critical in cellular responses such as apoptosis. They enhance mitochondrial outer membrane permeabilization (MOMP) through self-assembly in the mitochondrial outer membrane to form channels that release cytochrome c from intermembrane space (IMS) into the cytosol, triggering caspase-9 activation. However, the SMase involved in MOMP is yet to be identified. Here, we identified a mitochondrial Mg²⁺-independent SMase (mt-iSMase) from rat brain, which was purified 6,130-fold using a Percoll gradient, pulled down with biotinylated sphingomyelin, and subjected to Mono Q anion exchange. A single peak of mt-iSMase activity was eluted at a molecular mass of approximately 65 kDa using Superose 6 gel filtration. The purified enzyme showed optimal activity at pH of 6.5 and was inhibited by dithiothreitol and Mg²⁺, Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Fe²⁺, and Fe³⁺ ions. It was also inhibited by GW4869, which is a non-competitive inhibitor of Mg²⁺-dependent neutral SMase 2 (encoded by SMPD3), that protects against cytochrome c release-mediated cell death. Subfractionation experiments showed that mt-iSMase localizes in the IMS of the mitochondria, implying that mt-iSMase may play a critical role in generating ceramides for MOMP, cytochrome c release, and apoptosis. These data suggest that the purified enzyme in this study is a novel SMase.

• Journal of Life Science
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• v.26 no.3
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• pp.275-281
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• 2016

Glycan modification is important in pharmaceutical industry. Especially, sialic acid affects the bioactivity and stability of medicine. Milk of pig has been used as bioreactor to produce various pharmaceutical proteins. Therefore, it is necessary to modify the glycan chain in pig mammary grand. β-1,4-N-Acetylglucosaminyltransferase A (pMGAT4A) is one of the essential enzymes for increase of sialic acid content, but pig MGAT4A is unclear. In this study, the pMGAT4A was identified and characterized. The pMGAT4A has 1638 nucleotides encoding 535 amino acids and type II membrane topology, which is one of the common features in many glycosyltransferases. The gene was strongly expressed in liver and mammary gland, whereas was weakly expressed in small intestine, stomach and bladder. For functional test, HA-tagged MGAT4A was over-expressed in porcine kidney (PK-15) cell line. Forced expression of pMGAT4A gene was identified by qPCR, and we identified that pMGAT4A is located in Golgi complex by co- staining with HA antibody and BODIPY TR ceramide. In addition, we identified the increase of mannose-β-1,4-N-acetylglucosamine structure by ELISA and immunofluorescence using Datura stramonium agglutinin (DSA), which recognizes mannose-β-1,4-Nacetylglucosamine. Through the specific activity analysis, we showed that pMGAT4A modified bi-antennary to tri-antennary. This event affects sialic acid content. Therefore, we thought that over-expression of pMGAT4A will be necessary in pig mammary grand for improved medicine.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.11
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• pp.1611-1618
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• 2010

This study was conducted to evaluate the effects of Eriobotrya japonica and Saurus chinensis extracts and their fermented extracts on immune parameters in BALB/c mice treated with 1% 2,4-dinitrochlorobenzene (DNCB). The groups were Eriobotrya japonica extract (BI), Saurus chinensis extract (SA), mixture with E. japonica extract and S. chinensis extract (FB) and fermented mixture with E. japonica extract and S. chinensis extract (FA) and distilled water treated control. The level of serum immunoglobulin (Ig) E was decreased in FA compared to control group, but significant difference was not observed (p<0.05). The histamine and contents in FA and control group were $1.47{\pm}0.20$ ng/mL and $1.90{\pm}0.04$ ng/mL, respectively (p<0.05). Ceramide contents were significantly increased in FA compared to BI, SA, FB and control group (p<0.05). These results suggest that FA supplementation in the DNCB treated BALB/c mice affect anti-allergic activities positively, and may be used as functional material for suppression of atopy dermatitis in food industry.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.32 no.1 s.55
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• pp.45-51
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• 2006

In the field of makeup cosmetics, especially, powder-based foundations such as two-way cake, pact and face powder, the quality of which is known to be strongly influenced by the properties of powder, surface treatment technology is widely used as a method to improve the various characteristics of powder texture, wear properties, dispersion ability and so on. The two-way cake or pressed-powder foundation is one of the familiar makeup products in Asian market for deep covering and finishing purpose. In spite of the relent progress in surface modification method such as composition of powders with different characteristics and application of a diversity of coating ingredient (metal soap, amino acid, silicone and fluorine), this product possess a technical difficulty to enhance both of the adhesion power and spreadability on the skin in addition to potential claim of consumer about heavy or thick feeling. This article is covering the preparation and coating method of nano-vesicle that mimic the double-layered lipid lamellar structure existing between the corneocytes of the stratum corneum in the skin for the purpose of improving both of two important physical characteristic of two-way cake, spreadability and adhering force to skin, and obtining better affinity to skin. Nano-vesicle was prepared using the high-pressure emulsifying process of lecithin, pseudo ceramide, butylene glycol and tocopheryl acetate. This nano-sized emulsion was added to powder-dispersed aqueous phase together with bivalent metal salt solution and then the filtering and drying procedure was followed to yield the nano-vesicle coated powder. The amount of nano-vesicle coated on the powder was able to regulated by the concentration of metal salt and this novel powder showed the lower friction coefficient, more uniform condition of application and higher adhesive powder comparing with the alkyl silane treated powder from the test result of spreadability and wear properties using friction meter and air jet method. Two-wav cake containing newly developed coated powder with nano-vesicle showed the similar advantages in the frictional and adhesive characteristics.

• Journal of Veterinary Clinics
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• v.27 no.5
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• pp.522-532
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• 2010

The aims of the present study were to characterize the effect of glucocorticosteroids (GCs) on the normal canine skin and to evaluate the effect of a lipid mixture (LM), containing cholesterol, pseudoceramide, and free fatty acid, on the steroid-induced damaged skin of dogs. Five beagles were involved and the skin of the back of each dog was topically applied with four kinds of GCs twice daily for 28 days. LM was applied after that period of GCs application. Transepidermal water loss (TEWL), skin hydration, and skin pH were assessed during experimental periods and histopathological evaluation was performed. TEWL was significantly increased, with a maximum increase obtained on day 28 (p < 0.01). Skin pH was significantly decreased, with a maximum decrease obtained on day 28 (p < 0.01). Skin surface hydration was significantly increased on day 3, but values of skin hydration were progressively decreased and finally reached those of baseline. In histology, as results of steroid application, losses of keratin layers in the stratum corneum and edematous changes in the upper parts of dermis, and consequently, thickness of the epidermis and the stratum corneum were decreased. In addition, the numbers of hair follicles were markedly decreased in steroid control as compared to intact control. However, these skin atrophic changes were markedly inhibited by treatment of LM as compared with steroid control in the present study. Moreover, all biophysical parameters were reached to the baseline after LM treatment. These results showed that the topically applied GCs induced skin barrier impairment and a LM should be effective on repair of disturbed skin barrier function in dogs. Therefore, it is concluded that a LM tested in the present study is expected to treat the steroid-induced skin damages.

• Journal of Genetic Medicine
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• v.4 no.1
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• pp.45-52
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• 2007

Purpose : A simple, rapid, and highly sensitive analytical method for Gb3 in plasma was developed without labor-ex tensive pre-treatment by electrospray ionization MS/ MS (ESI-MS/MS). Measurement of globotriaosy lceramide (Gb3, ceramide trihex oside) in plasma has clinical importance for monitoring after enzyme replacement therapy in Fabry disease patients. The disease is an X-linked lipid storage disorder that results from a deficiency of the enzyme ${\alpha}$-galactosidase A (${\alpha}$-Gal A). The lack of ${\alpha}$-Gal A causes an intracellular accumulation of glycosphingolipids, mainly Gb3. Methods : Only simple 50-fold dilution of plasma is necessary for the extraction and isolation of Gb3 in plasma. Gb3 in diluted plasma was dissolved in dioxane containing C17:0 Gb3 as an internal standard. After centrifugation it was directly injected and analyzed through guard column by in combination with multiple reaction monitoring mode of ESI-MS/MS. Results : Eight isoforms of Gb3 were completely resolved from plasma matrix. C16:0 Gb3 occupied 50% of total Gb3 as a major component in plasma. Linear relationship for Gb3 isoforms w as found in the range of 0.001-1.0 ${\mu}g$/mL. The limit of detection (S/N=3) was 0.001 ${\mu}g$/mL and limit of quantification was 0.01 ${\mu}g$/mL for C16:0 Gb3 with acceptable precision and accuracy. Correlation coefficient of calibration curves for 8 Gb3 isoforms ranged from 0.9678 to 0.9982. Conclusion : This quantitative method developed could be useful for rapid and sensitive 1st line Fabry disease screening, monitoring and/or diagnostic tool for Fabry disease.