• 한국산학기술학회논문지
• /
• 제10권3호
• /
• pp.648-653
• /
• 2009

p62는 산화스트레스가 주요 원인 중 하나인 퇴행성 뇌질환과 연관된 엉긴 물질(aggregate)의 주요 구성성분이다. 퇴행성 뇌질환과 관련된 것으로 알려진 hydroxydopamin이나 $C_2-ceramide$를 신경모세포종 세포주인 SH-SY5Y에 처리하였을 때 p62의 발현이 유도되며 시간이 지날수록 그 양이 증가되었다. 또한 p62를 과발현시키면 ceramide에 의한 SH-SY5Y 세포의 사멸이 지연되었다. 이 과정에서 P62는 시간이 경과됨에 따라 침전화되며 절단되었다. 이 결과로 p62의 신경보호효과와 여러 종류의 퇴행성 뇌질환에서 p62가 엉긴 물질에서 발견되는 이유를 추측할 수 있다.

• Journal of Microbiology and Biotechnology
• /
• 제20권2호
• /
• pp.356-362
• /
• 2010

Ceramide is important not only for the maintenance of the barrier function of the skin but also for the water-binding capacity of the stratum corneum. Although the exact role of ceramide in the human skin is not fully understood, ceramide has become a widely used ingredient in cosmetic and pharmaceutical industries. Compared with other microorganisms, yeast is more suitable for the production of ceramide because yeast grows fast and is non-toxic. However, production of ceramide from yeast has not been widely studied and most work in this area has been carried out using Saccharomyces cerevisiae. Regulating the genes that are involved in sphingolipid synthesis is necessary to increase ceramide production. In this study, we investigated the effect of the genes involved in the synthesis of ceramide, lcb1, lcb2, tsc10, lac1, lag1, and sur2, on ceramide production levels. The genes were cloned into pYES2 high copy number vectors. S. cerevisiae was cultivated on YPDG medium at $30^{\circ}C$. Ceramide was purified from the cell extracts by solvent extraction and the ceramide content was analyzed by HPLC using ELSD. The maximum production of ceramide (9.8 mg ceramide/g cell) was obtained when the tsc10 gene was amplified by the pYES2 vector. Real-time RT-PCR analysis showed that the increase in ceramide content was proportional to the increase in the tsc10 gene expression level, which was 4.56 times higher than that of the control strain.

• 약학회지
• /
• 제41권1호
• /
• pp.94-98
• /
• 1997

Ceramide. a product of sphingomyelin hydrolysis, has been proposed as a lipid second messenger mediating antiproliferative activation. In this study, we examined the role of the cell cycle-related proteins in the ceramide-mediated growth suppression. Treatment of U-937 cells with C$_2$-ceramide(N-acetylsphingosine) resulted in growth suppression in a time- and concentration dependent manner. Ceramide induced concentration dependent dephosphorylation of retinoblastoma gene product (Rb). Rb remains hypophosphorylated in synchronized cells even after serum stimulation in the presence of ceramide. Ceramide decreased the expression of cyclin D$_1$ and cyclin E levels. These results suggest that antiproliferative effect of ceramide is associated with hypophosphorylation of Rb and decreased expression of cyclin D1 and cyclin E.

• Biomolecules & Therapeutics
• /
• 제27권2호
• /
• pp.193-200
• /
• 2019

Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z $588.6{\rightarrow}264.4$ for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625-160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.

• Molecular & Cellular Toxicology
• /
• 제3권4호
• /
• pp.273-281
• /
• 2007

Ceramide is involved in cell death as a lipid mediator of stress responses. In this study, we developed an improved method of ceramide quantification based on added synthetic ceramide and thin layer chromatography (TLC) separation, and applied to biological samples. Lipids were extracted from samples spiked with N-oleoyl-D-erythro-sphingosine ($C_{17}$ ceramide) as an internal standard. Ceramide was resolved by TLC, complexed with fatty-acidfree bovine serum albumin (BSA), and deacylated by ceramidase (CDase). The released sphingosine was derivatized with o-phthalaldehyde (OPA) and measured by high performance liquid chromatography (HPLC). The limit of detection for ceramide was about 1-2 pmol and the lower limit of quantification was 5 pmol. Ceramide recovery was approximately 86-93%. Ceramide concentrations were determined in biological samples including cultured cells, mouse tissues, and mouse and human plasma. TLC separation of ceramide provides HPLC chromatogram with a clean background without any interfering peaks and the enhanced solubility of ceramide by BSAceramide complex leads to the increased deacylation of ceramide. The use of an internal standard for the determination of ceramide concentration in these samples provides an accurate and reproducible analytical method, and this method can be applicable to diverse biological samples.

• BMB Reports
• /
• 제37권2호
• /
• pp.192-198
• /
• 2004

The hepatitis C virus (HCV) core protein is believed to be one of viral proteins that are capable of preventing virus-infected cell death upon various stimuli. But, the effect of the HCV core protein on apoptosis that is induced by various stimuli is contradictory. We examined the possibility that the HCV core protein affects the ceramide-induced cell death in cells expressing the HCV core protein through the sphingomyelin pathway. Cell death that is induced by $C^2$-ceramide and bacterial sphingomyelinase was analyzed in 293 cells that constitutively expressed the HCV core protein and compared with 293 cells that were stably transfected only with the expression vector. The HCV core protein inhibited the cell death that was induced by these reagents. The protective effects of the HCV core protein on ceramide-induced cell death were reflected by the reduced expression of $p21^{WAF1/Cip1/Sid1}$ and the sustained expression of the Bcl-2 protein in the HCV core-expressing cells with respect to the vector-transfected cells. These results suggest that the HCV core protein in 293 cells plays a role in the modulation of the apoptotic response that is induced by ceramide. Also, the ability of the HCV core protein to suppress apoptosis might have important implications in understanding the pathogenesis of the HCV infection.

• The Korean Journal of Physiology and Pharmacology
• /
• 제6권6호
• /
• pp.281-286
• /
• 2002

To understand the cytotoxic mechanism of $MPP^+,$ we examined the involvement of ceramide in $MPP^+-induced$ cytotoxicity to human neuroblastoma SH-SY5Y cells. When SH-SY5Y cells were exposed to $MPP^+,\;MPP^+$ induced dose-dependent cytotoxicity accompanied by 2-fold elevation of intracellular ceramide levels in SH-SY5Y cells. Three methods were used to test the hypothesis that the elevated intracellular ceramide is related to $MPP^+-induced$ cytotoxicity: $C_2-ceramide$ was directly applied to cells, sphingomyelinase (SMase) was exogenously added, and oleoylethanolamine (OE) was used to inhibit degradation of ceramide. Furthermore, inhibition of ceramide-activated protein phosphatase (CAPP), the effector of ceramide, using okadaic acid (OA) attenuated cell death but treatment of fumonisin $B_1,$ the ceramide synthase inhibitor, did not alter the cytotoxic effect of $MPP^+.$ Based on these, we suggest that the elevation of intracellular ceramide is one of the important mediators in $MPP^+-induced$ cell death.

• 한국환경성돌연변이발암원학회:학술대회논문집
• /
• 한국환경성돌연변이발암원학회 2002년도 Molecular and Cellular Response to Toxic Substances
• /
• pp.144-144
• /
• 2002

• 대한한방소아과학회지
• /
• 제35권3호
• /
• pp.128-137
• /
• 2021

Objective The purpose of this study was to confirm the effectiveness of coptidis rhizoma extract-ceramide complex on skin barrier, transepidermal water loss (TEWL) and pH reduction, and inflammation of the skin. Methods Coptidis rhizoma extract-ceramide complex was applied in 6-week-old Balb/C mice after dermatitis was induced. To confirm the skin condition changes, TEWL and pH were observed, and filaggrin in the stratum corneum of the skin was observed. Kallikrein-related peptidase (KLK) 7, Protease activated receptor (PAR)-2, Thymic stromal lymphopoietin (TSLP), and IL-4 were observed in the stratum corneum to confirm the changes in the inflammatory response. Results Filaggrin positive reaction was increased in the experiment group compared to the control group. TEWL and pH were lower in the experiment group compared to the control group. KLK7, PAR2, TSLP, and IL-4 positive responses were decreased in the experiment group compared to the control group. Conclusions It was confirmed that the coptidis rhizoma extract-ceramide complex can relieve the inflammatory response of atopic dermatitis by restoring the skin lipid barrier damage.

• Molecules and Cells
• /
• 제19권2호
• /
• pp.185-190
• /
• 2005

The chicken oviduct is a dynamic organ that produces secretory proteins such as ovalbumin and its cells undergo cell proliferation and differentiation. There has been no study of the cellular mechanism involved in cell death in the chicken oviduct. Therefore, this study has focused on the study of apoptosis in primary oviduct cells. Because ceramide is known to activate apoptosis in tumor cells and is produced in the oviduct, we used an exogenous ceramide analog to induce cell death. The viability of ceramide-treated chicken oviduct cells decreased in a dose-dependent manner and apoptotic cells were detected by staining with annexin V. The expression of apoptosis-related genes was assessed by RT-PCR and bcl-2 mRNA was found to decrease after exposure to ceramide while Bcl-x mRNA increased 12 h post-treatment. In addition, caspase-3 was expressed strongly in the early stages of apoptosis, while caspase-1 and -9 transcripts increased at later times. We conclude that ceramide induces apoptosis in oviduct-derived primary cells via a caspase- and bcl-2-dependent pathway.