• Toxicological Research
• /
• 제11권1호
• /
• pp.1-8
• /
• 1995

Fumonisins are a family of mycotoxins produced by the fungus Fusarium moniliforme which is a common contaminant in corn. Fumonisins are potent inhibitors of sphingosine and sphinganine N-acyltransferase (ceramide synthase), key enzymes in sphingolipid metabolism. The purpose of this study was to provide the evidence that the elevated levels of free sphingoid bases (primarily sphinganine) and depletion of complex sphingolipids were closely related to the inhibition of cell growth in LLC-$PK_1$ cells exposed to fumonisin $B_1$$(\leq 35 {\mu}M)$. Concentrations of fumonisin $B_1$ between 10 and $35 {\mu}M$ were known to inhibit cell growth without cytotoxicity in $LLC-PK_1$ cells (Yoo et al. Toxicol. Appl. Pharmacol. 114, 9-15, 1992). Cells exposed to 35$\mu M$ fumonisin B$_1$ for 48 and 72 hr developed a fibroblast-like (elongated and spindle-shaped) appearance and were less confluent than normal cells. At between 24 and 48 hr after exposure to fumonisin $B_1$ cells were beginning to show the inhibition of cell growth and at 72 hr the number of viable cells in fumonisin-treated cultures was about 50% of concurrent control cultures. During the 24 hr lag period preceding inhibition of cell growth, the free sphinganine levels in cells exposed to $35 {\mu}M$ fumonisin $B_1$ were highly elevated (approximately 230 fold higher than normal cells). The elevated levels of free sphinganine were $435\pm14$$pmoles/{10^6}$ cells at 48 hr and approximately TEX>$333\pm11$$pmoles/{10^6}$ cells in cells exposed to $35{\mu}M$ fumonisin$B_1$ at 72 hr, while the levels of free sphinganine in normal cells were less than 2$pmoles/{10^6}$ cells. Under the same condition, depletion of intracellular complex sphingolipids as a consequence of fumonisin inhibition of de novo sphingolipid biosynthesis and turnover pathway was appeared. Content of free sphingold bases in dividing cells was more elevated than in confluent cells at 24-48 hr after cells were exposed to $20{\mu}M$ fumonisin $B_1$. The dividing cells were showing the inhibition of cell growth at 48-72 hr and $20{\mu}M$ fumonisin $B_1$. The results of this study support the hypothesis that the inhibition of cell growth is very well related to the disruption of sphingolipid metabolism in $LLC-PK_1$ cells.

• Biomolecules & Therapeutics
• /
• 제19권1호
• /
• pp.134-139
• /
• 2011

Sphingosine kinase (SPHK) has a central role to control cell death and cell proliferation, which is suggested as a sphingolipid rheostat by regulating the levels between ceramide and sphingosine 1-phosphate (S1P). Therefore, physiological regulators of SPHK will be a good candidate to develop a new targeted drug. For this purpose, a series of synthetic pseudoceramides were tested by SPHK assay either cell-based or cell-free system. K10PC-5 strongly inhibited SPHK, while K6PC-5 activated SPHK in cell-free system. Specifically, K6PC-5 activated SPHK under the co-treatment with $50\;{\mu}M$ dimethylsphingosine (DMS), a SPHK inhibitor. Collectively, we developed a simple SPHK assay system to find SPHK regulatory pseudoceramide compounds, K10PC-5 and K6PC-5 which may be useful to cancer treatment or immune regulation like FTY720, a synthetic sphingolipid mimetic compound.

• Biomolecules & Therapeutics
• /
• 제16권1호
• /
• pp.34-39
• /
• 2008

Oxidative stress may represent a common link between chronic liver damage and hepatic fibrosis. In the present study, we investigated activity changes of sphingomyelin catabolic enzymes, such as sphingomyelinases and ceramidases by using dimethylnitrosamine (DMN)-treated Sprague-Dawley (SD) male rats hepatic fibrosis model as a hepatic fibrosis model. Twenty rats divided into five groups received: (1) saline; (2) DMN for 1 week, (3) DMN for 2 weeks, (4) DMN for 3 weeks, and (5) DMN for 4 weeks by intraperitoneally 10 mg/kg of body weight for three consecutive days a week. Activities of acidic and neutral sphingomyelinases and acidic, neutral and alkaline ceramidases were measured in the liver and kidney from DMN-treated rats. We found increased ceramidase activities from 2-week and/or 3-week DMN treated rat livers compared to control rat liver. Acidic sphingomyelinase and alkaline ceramidase activities were significantly increased in 3-week DMN-treated rat kidneys compared to control rat kidney. Therefore, sphingolipid metabolizing enzymes and sphingolipid metabolites are supposed to be involved in liver fibrosis, although further investigation is necessary to elucidate meanings of sphingolipids during the liver fibrosis

• Applied Biological Chemistry
• /
• 제50권1호
• /
• pp.72-76
• /
• 2007

대표적인 스핑고지질인 글리코실세라미드의 생리적 기능을 조사하기 위하여 쌀겨 추출물로부터 cerebroside를 분리하였다. 정제하지 않은 글리코실세라미드를 flash system 칼럼으로 분리한 후 ELSD를 검출기로 사용하여 순상 HPLC로 정제하였다. 클로로포름 : 메탄올 : 증류수(99:11:1, v/v/v)을 용출용매로 사용하여 주요 cerebroside를 얻을 수 있었고, MALDI/TOF-MS, FAB-MS, GC, 600MHz $^1$H-NMR로 구조를 분석하였다. 구성당은 글루코오스였고 cerebroside의 구성 지방산은 2-hydroxy-arachidic산이었다. 장쇄 염기는 sphinga-4,8-dienine이었다.

• BMB Reports
• /
• 제55권12호
• /
• pp.621-626
• /
• 2022

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by the degeneration of motor neurons in the spinal cord. Main symptoms are manifested as weakness, muscle loss, and muscle atrophy. Some studies have reported that alterations in sphingolipid metabolism may be intimately related to neurodegenerative diseases, including ALS. Acid sphingomyelinase (ASM), a sphingolipid-metabolizing enzyme, is considered an important mediator of neurodegenerative diseases. Herein, we show that ASM activity increases in samples from patients with ALS and in a mouse model. Moreover, genetic inhibition of ASM improves motor function impairment and spinal neuronal loss in an ALS mouse model. Therefore, these results suggest the role of ASM as a potentially effective target and ASM inhibition may be a possible therapeutic approach for ALS.

• 대한약학회:학술대회논문집
• /
• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.1
• /
• pp.162.1-162.1
• /
• 2003

The purpose of this study was to determine the possibility of sphingolipid as a diagnostic marker for Myocardiac Infarction(MI), atherosclerosis-related cardiovascular disease. Sphingolipids are known to playa role in the occurrence of atherosclerosis in human blood vessels. Platelet-poor plasma(PPP) and washed platelets were prepared from healthy volunteers and MI patients, and sphingolipids analyzed. (omitted)

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2000년도 춘계학술발표대회
• /
• pp.588-591
• /
• 2000

Sphingoliped 대사의 율속효소인 serine palmitoyl transferase(SPT)와 acid ceramidase의 연구를 위하여 인간의 SPTII 유전자와 acid ceramidase 유전자의 5‘-upstream region을 얻었다. 사람의 대장암세포인 HT29 cell로부터 genomic DNA를 얻고 GenomeWalker kit를 이용하였으며 2690bp의 SPTII promoter와 2028bp 및 1034bp의 acid ceramidase promoter의 fragment들을 얻을 수 있었다. 이들 DNA 조각들을 T7Blue vector에 subcloning하여 sequencing하였으며 이들이 사람의 SPTII 및 acid ceramidase gene의 5’-upstream region임을 확인하였다. 동물세포에서의 promoter activity를 측정하기위하여 firefly luciferase를 reporter gene으로 하는 pGL2-enhancer vector와 pGL2-basic vector에 subcloning하였으며 pRL-TK vctor와 함께 HT29 cell 및 HepG2 cell에 cotransfection 시킨 후 luciferaseg활성을 측정한 결과 같은 양의 DNA로는 사람의 SPTII promoter와 acid ceramidase promoter는 pRL-TK에 비하여 transfection efficiency가 아주 낮았으며 promoter 연구를 위하여는 pRL-TK vector의 양을 1/100으로 줄이는 것이 적당하였으며 HT29 cell보다는 HepG2 cell에 더 높은 발현율을 보였다.

• 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.

• BMB Reports
• /
• 제52권3호
• /
• pp.220-225
• /
• 2019

We have identified a mechanism to diminish the proliferative capacity of cells during cell expansion using human adipose-derived stromal cells (hAD-SCs) as a model of replicative senescence. hAD-SCs of high-passage numbers exhibited a reduced proliferative capacity with accelerated cellular senescence. Levels of key bioactive sphingolipids were significantly increased in these senescent hAD-SCs. Notably, the transcription of sphingosine kinase 1 (SPHK1) was down-regulated in hAD-SCs at high-passage numbers. SPHK1 knockdown as well as inhibition of its enzymatic activity impeded the proliferation of hAD-SCs, with concomitant induction of cellular senescence and accumulation of sphingolipids, as seen in high-passage cells. SPHK1 knockdown-accelerated cellular senescence was attenuated by co-treatment with sphingosine-1-phosphate and an inhibitor of ceramide synthesis, fumonisin $B_1$, but not by treatment with either one alone. Together, these results suggest that transcriptional down-regulation of SPHK1 is a critical inducer of altered sphingolipid profiles and enhances replicative senescence during multiple rounds of cell division.

• 대한약학회:학술대회논문집
• /
• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.1
• /
• pp.288.3-289
• /
• 2003

Sphingolipid species are important second messengers due to their role in the mitogenesis, differentiation and apoptosis. We developed a new column liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS) in combination with multiple reaction monitoring (MRM) method for the rapid, simultaneous and quantitative determination of unambiguous detecting sphingolipids in cell culture of human cancer cells (HL-60). (omitted)