1.Effect of extracts from Radix Trichosanthis on the expression of HBsAg and HBeAg in HepG2.2.15 cells.
Jia CHEN ; Zhaofa XU ; Hongtao OUYANG ; Minyuan PENG ; Ting WU ; Jing LIU ; Yanping LIU ; Huiwen YAN
Journal of Central South University(Medical Sciences) 2012;37(1):38-41
OBJECTIVE:
To investigate the effect of extracts with water and alcohol from Radix Trichosanthis on the cell survival and the expression of hepatitis B surface antigen (HBsAg) and hepatitis B e-antigen (HBeAg) in HepG2.2.15 cell supernatant.
METHODS:
The cell survival rate of HepG2.2.15 cells was detected by MTT assay. The HBsAg and HBeAg in HepG 2.2.15 cell supernatant were evaluated by enzyme linked immunosorbent assay.
RESULTS:
The water and alcohol soluble extracts from Radix Trichosanthis significantly inhibited the levels of HBsAg and HBeAg in HepG2.2.15 cells in a time-and-concentration-dependent manner. However, the therapeutic index of extracts with water from Radix Trichosanthis was better than that in the alcohol group.
CONCLUSION
The activity of water-soluble extract from Radix Trichosanthis is stronger on anti-hepatitis B virus than that of the alcohol-soluble extract.
Antiviral Agents
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pharmacology
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Drugs, Chinese Herbal
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classification
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pharmacology
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Hep G2 Cells
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Hepatitis B Surface Antigens
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biosynthesis
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Hepatitis B e Antigens
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biosynthesis
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Hepatitis B virus
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drug effects
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Humans
2.Mechanism of the Rpn13-induced activation of Uch37.
Lianying JIAO ; Songying OUYANG ; Neil SHAW ; Gaojie SONG ; Yingang FENG ; Fengfeng NIU ; Weicheng QIU ; Hongtao ZHU ; Li-Wei HUNG ; Xiaobing ZUO ; V ELEONORA SHTYKOVA ; Ping ZHU ; Yu-Hui DONG ; Ruxiang XU ; Zhi-Jie LIU
Protein & Cell 2014;5(8):616-630
Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other's ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37Δ(Hb,Hc,KEKE), a truncation removal of the C-terminal extension region (residues 256-329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270-407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.
Binding Sites
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Catalytic Domain
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Chromatography, Gel
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Crystallography, X-Ray
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Humans
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Membrane Glycoproteins
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chemistry
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genetics
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metabolism
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Nuclear Magnetic Resonance, Biomolecular
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Protein Binding
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Protein Conformation
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Protein Multimerization
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Scattering, Small Angle
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Ubiquitin Thiolesterase
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chemistry
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genetics
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metabolism
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Ultracentrifugation