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Secretion of granular glands from the skin of amphibians is a fascinating resource of active substances, particularly for cancer therapy in clinical practice of Traditional Chinese Medicine. A variety of anti‑tumor peptides have been isolated from different toads and frogs; however, no anti‑tumor peptides are reported in toad venom of Bufo gargarizans. Firstly, soluble fraction from fresh toad venom (FTV) was compared with that from dried toad venom (DTV), using HPLC analysis. It was revealed that FTV has a different HPLC chromatography compared with DTV. Soluble fraction of FTV is more toxic to SH‑SY5Y cells than that of DTV, as evaluated by MTT assay. Secondly, it was identified that protein components from soluble fractions of FTV and DTV possess different patterns by SDS‑PAGE analysis, and proteins from FTV are also more toxic than that from DTV. Thirdly, an immobilized basic fibroblast growth factor (bFGF) affinity column was used to isolate bFGF‑binding components from soluble fraction of FTV, and it was identified that bFGF‑binding components prohibited bFGF‑dependent neurite growth of SH‑SY5Y cells. Finally, it was identified that bFGF‑binding components activated apoptosis via upregulation of caspase‑3 and caspase‑8 expression in SH‑SY5Y cells. These data suggest that FTV contains active components that interact with bFGF and activate apoptosis in SH‑SY5Y cells. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-6 is a cytokine produced mainly by microglia and astrocytes and plays a pleiotropic role in the central nervous system. In this study, we cloned rat lL-6 cDNA into an enhanced green fluorescent protein (EGFP) or a red fluorescent protein (DsRed2) vector and rat 78-kDa glucose-regulated protein (GRP78) cDNA into an EGFP vector to construct IL-6-EGFP, lL-6-DsRed2, and GRP78-EGFP chimeras for the investigation of the mechanism of IL-6 secretion from astrocytes. The data showed that constructed IL-6-EGFP and IL-6-DsRed2 chimeras retained the secretory property, and the secretion of IL-6-EGFP from astrocytes could be attenuated by GRP78 depletion with double- stranded RNA interference. Coexpression of IL-6-DsRed2 and dysfunctional GRP78-EGFP abolished IL-6-DsRed2 secretion, and two chimeric proteins colocalized inside living astrocytes. Coimmunoprecipitation analysis indicated that IL-6 and GRP78 resided in the same complex. The data further revealed that IL-6-EGFP secretion from astrocytes was blocked by the heavy metal lead (Pb) in a concentration-dependent manner. Analysis of the Pb interaction with protein on a Pb-affinity column demonstrated that Pb bound to GRP78 but failed to bind to IL-6. Therefore, these data suggest that IL-6-EGFP or IL-6-DsRed2 chimeras can be used as imaging probes to study IL-6 secretion from living cells, that GRP78 is involved in IL-6 secretion from astrocytes, and that Pb can block IL-6 secretion from astrocytes via targeting GRP78. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-6 is a cytokine produced mainly by microglia and astrocytes and plays a pleiotropic role in the central nervous system. In this study, we cloned rat IL-6 cDNA into an enhanced green fluorescent protein (EGFP) or a red fluorescent protein (DsRed2) vector and rat 78-kDa glucose-regulated protein (GRP78) cDNA into an EGFP vector to construct IL-6-EGFP, IL-6-DsRed2, and GRP78-EGFP chimeras for the investigation of the mechanism of IL-6 secretion from astrocytes. The data showed that constructed IL-6-EGFP and IL-6-DsRed2 chimeras retained the secretory property, and the secretion of IL-6-EGFP from astrocytes could be attenuated by GRP78 depletion with double-stranded RNA interference. Coexpression of IL-6-DsRed2 and dysfunctional GRP78-EGFP abolished IL-6-DsRed2 secretion, and two chimeric proteins colocalized inside living astrocytes. Coimmunoprecipitation analysis indicated that IL-6 and GRP78 resided in the same complex. The data further revealed that IL-6-EGFP secretion from astrocytes was blocked by the heavy metal lead (Pb) in a concentration-dependent manner. Analysis of the Pb interaction with protein on a Pb-affinity column demonstrated that Pb bound to GRP78 but failed to bind to IL-6. Therefore, these data suggest that IL-6-EGFP or IL-6-DsRed2 chimeras can be used as imaging probes to study IL-6 secretion from living cells, that GRP78 is involved in IL-6 secretion from astrocytes, and that Pb can block IL-6 secretion from astrocytes via targeting GRP78.