• BMB Reports
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• 제30권5호
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• pp.370-377
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• 1997

A novel assay method is described for rapid quantitation of reaction rate of sterol ${\Delta}^7$-reductase (${\Delta}^7$-SR) which catalyzes reduction of the ${\Delta}^7$-double bond of sterols. Of six different organ tissues-liver, small intestine, brain, lung, kidney, and testis-. ${\Delta}^7$-SR activity was detected only in liver (2.30 nmol/min/mg protein) and testis (0.11 nmol/min/mg protein). Using a newly developed method which employs diet-induced enzyme proteins and ergosterol as substrate, we assessed both kinetics ($K_m=210\;{\mu}M$, $V_{max}=1.93\;nmol/min/mg$) and inhibition of the rat hepatic ${\Delta}^7$-SR against well-studied cholesterol lowering agents such as triparanol ($IC_{50}=16\;{\mu}M$). 3-$\beta$-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A) ($IC_{50}=5.2\;{\mu}M$), and trans-1.4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride (AY-9944) ($IC_{50}=0.25\;{\mu}M$). Of the three well-known AY-9944-sensitive cholesterogenic enzymes (i.e., ${\Delta}^7$-SR, sterol ${\Delta}^8$-isomerase, and sterol ${\Delta}^14$-reductase). ${\Delta}^7$-SR was found to be the most sensitive enzyme with a noncompetitive inhibition of this compound ($K_i=0.109\;{\mu}M$). Substrate specificity studies of the microsomal ${\Delta}^7$-SR indicate that the relative reaction rate for 7-dehydrocholesterol and ergosterol are 5.6-fold and 1.6-fold higher than that for lathosterol. ${\Delta}^7$-SR activity was also modulated by feeding rats a diet supplemented with 0.5% ergosterol (>2.6-fold) in addition to 5.0% cholestyramine plus 0.1% lovastatin ($\simeq$5.0-fold). Finally, microsomal ${\Delta}^7$-SR was solubilized by 1.5% 3-[3-(cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and enriched on PEG (0~10%) precipitation, which should be suitable for further purification of the enzyme.

• Asian-Australasian Journal of Animal Sciences
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• 제22권3호
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• pp.319-327
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• 2009

Follicular fluid meiosis-activating sterol (FF-MAS) has been suggested as a positive factor which could improve the oocyte quality and subsequent embryo development after in vitro fertilization. However, FF-MAS is a highly lipophilic substance and is hard to detect in studying the relationship between MAS and quality of oocyte maturation. The present study focused on the expression of lanosterol 14${\alpha}$-demethylase (LDM), a key enzyme that converts lanosterol to FF-MAS, on mouse oocyte maturation and its potency on development. LDM expression was strong in gonadotropin-primed germinal vesicle stage oocytes, weak after germinal vesicle breakdown (GVBD), and then strong in MII stage oocytes. The LDM-specific inhibitor azalanstat significantly inhibited oocyte fertilization (from 79.4% to 68.3%, p<0.05). Also, azalanstat (5 to 50 ${\mu}M$) decreased the percentage of blastocyst development dosedependently (from 78.7% to 23.4%, p<0.05). The specific inhibition of sterol ${\Delta}14$-reductase and ${\Delta}7$-reductase by AY9944 accumulates FF-MAS and could increase blastocyst development rates. Additionally, in the AY9944 group, the rate of inner cell mass (ICM)/ total cells was similar to that of in vivo development, but the rate was significantly decreased in azalanstat treatment. In conclusion, LDM, the key enzyme of FF-MAS production, may play an important role in fertilization and early development of the mouse embryo, especially in vitro.

• BMB Reports
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• 제31권6호
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• pp.565-571
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• 1998

The expression of the endogenous human apolipoprotein(apo)E gene was significantly induced when HepG2 cells were treated with exogenous 25-hydroxy-cholesterol. This sterol-mediated apoE gene upregulation appears to require the participation of a positive element for the apoE gene transcription (PET) ( -169/ -140), a core sequence of upstream regulatory element (URE)1 enhancer of the human apoE gene. This PET was renamed as sterol regulatory element (SRE)4 based on its new role as a sensor for the level of intracellular sterol. Furthermore, a gel mobility shift analysis showed that binding activity of the SRE4 binding protein (BP) obtained from HepG2 cells was induced by sterol treatment, while that from either MCF7 or BT20 cells remained unchanged. Binding activity of SRE4BP was also induced in mouse macrophage cells, J774A.1, by sterol treatment, but it was drastically reduced when cells were subjected to treatment of AY-9944, a potent inhibitor for sterol synthesis. However, binding activity of Spl, which is a co-binding protein to the SRE4 region, remained the same in either condition, suggesting that SRE4BP (formally known as PETBP) may be mainly responsible for the sterol-mediated regulation of the apoE gene expression. Deletion analysis of the core binding site of SRE4BP by gel mobility shift assays showed that the minimal sequence of the SRE4BP binding appears to reside between -157 and -140, confirming the identity of SRE4 with the previously determined core sequence of URE1.

• Biomolecules & Therapeutics
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• 제18권3호
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• pp.262-270
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• 2010

CD95 receptor is a member of tumor necrosis factor receptor family that mediates apoptosis in many cell types when bound by CD95 ligand or cross-linked by agonistic anti-CD95 antibodies. To determine the role of neutral sphingomyelinase (nSMase) on CD95-mediatd apoptosis, human Jurkat T lymphocytes were exposed to recombinant human CD95 ligand. Treatment with CD95 ligand induced cell death in a concentration and time-dependent manner. CD95-induced cell death was suppressed by inhibitors of SMase such as AY9944 or desipramine. Transfection with human nSMase1 siRNA plasmid into CD95 ligand-treated cells significantly prevented CD95-mediated cell death. CD95-mediated elevation of intracellular ceramide level detected by FACS analysis with anti-ceramide antibody was also decreased by nSMase1 siRNA. Knock-down of nSMase1 expression also blocked cytochrome c release into cytosol and caspase-3 cleavage in CD95-treated cells. Taken together, these results suggest that nSMase1 may play an important role in CD95-mediated apoptotic cell death in Jurkat T cells.