malachite-green-assay-for-discovery-heat-shock-protein-90

Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors

Heat shock protein 90 (Hsp90) is a promising anticancer target because of its chaperoning effect on multiple oncogenic proteins. The activity of Hsp90 is ...

Korea Institute of Science and Technology (KIST)

Determining the structure of REPI, an immunostimulatory polysaccharide fraction from radish leaves, is an important health objective. Herein, we show that REP-I contains nine different monosaccharides, including GalA (22.2%), Gal (32.6%), Ara (27.5%), and Rha (10.2%) as main sugars. REP-I was also reacted with β-glucosyl Yariv reagent (29.8%), suggesting the presence of the arabino-β-3,6-galactan. Furthermore, methylated-product analysis revealed that REP-I contains 13 different glycosyl linkages, including 4-linked GalpA (21.0%), 2,4-linked Rhap (7.0%), 4-linked Galp (5.8%), 5-linked Araf (10.1%), and 3,6-linked Galp (7.9%), which are characteristic of RG-I. Microstructural information was obtained by sequential degradation using four linkage-specific glycosylases and β-elimination, with fragments analyzed on the basis of sugar composition, methylation, and MS/MS spectra. The results show that the immunostimulatory activity of REP-I is possibly due to the structure of RG-I, which is composed of a main chain with repeating [→2)-Rhap-(1 → 4)-GalpA-(1→] linkage units and three side-chains: a branched α(1 → 5)arabinan, a β(1 → 4)galactan, and arabino-β-3,6-galactan, which are branched at the C(O)4 position of each Rha residue in the REP-I main chain.

Clarification of the structural features of Rhamnogalacturonan-I type polysaccharide purified from radish leaves.

In this study, the intracellular signaling pathways involved in macrophage activation through the RG-I-type polysaccharide (REP-I) purified from radish leaves were elucidated. The gene expression and secretion of immune-related factors such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and nitrogen oxide (NO) from macrophages were enhanced by the addition of REP-I. Moreover, immunoblotting and immunocytochemistry analyses indicated that REP-I dose-dependently phosphorylated the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways. An investigation using different inhibitors revealed that the effect of REP-I on NO secretion was mostly promoted by c-Jun N-terminal kinase (JNK) and NF-κB. Furthermore, the secretion of IL-6 was mostly induced via extracellular-signal-regulated kinase (ERK), JNK, and NF-κB. TNF-α secretion was mostly induced via NF-κB. In contrast, an investigation using anti-pattern recognition receptor (PRR) antibodies revealed that the effect of REP-I on the secretion of NO was mostly related with dectin-1, scavenger receptor (SR), toll-like receptor (TLR)2, TLR4, CD14, and CD11b. Furthermore, the secretion of IL-6 was mostly involved with SR, and the secretion of TNF-α was mostly relevance to TLR2. In conclusion, it is affirmed that immunostimulatory activation of macrophage of REP-I purified from radish leaves was deeply associated with several PRR and phosphorylating MAPK and NF-κB.

Immunostimulating and intracellular signaling pathways mechanism on macrophage of rhamnogalacturonan-I type polysaccharide purified from radish leaves.

SueJung Lee

Gangneung Institute of Natural Products

Korea Institute of Science and Technology (KIST)

Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors

SueJung Lee

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Korea Institute of Science and Technology (KIST)

Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors

Gangneung Institute of Natural Products