Hybridomas secreting a monoclonal antibody (mAb) against the herbicide chlorimuron-ethyl (CE) were produced by fusing the mouse myeloma cell line (SP2/0) with splenocytes from a mouse immunized against the conjugate of the sulfonamide moiety of CE and bovine serum albumin (BSA). The mAb, designated 1F5C5A10, had very weak affinity with metsulfuron, ethametsulfuron, pyrazosulfuron, bensulfuron, and chlorsulfuron. Two mAb-based indirect competitive enzyme-linked immunosorbent assays (icELISA) were developed. A conventional icELISA (icELISA-I) showed a concentration of half-maximum inhibition (IC(50)) of 11.6 ng/mL with a dynamic range of 1.6-84 ng/mL. A simplified icELISA (icELISA-II) had an IC(50) of 28.7 ng/mL and a dynamic range of 2.2-372 ng/mL. The two assays were tested on spiked water and soil samples. CE (1-500 ng/mL) fortified in water samples could be analyzed directly without any sample preparation by both immunoassays with an average recovery between 74 and 114%. icELISA-II, but not icELISA-I, was able to accurately analyze the herbicide residues in the crude soil extracts with recoveries between 99 and 129% without obvious matrix effects due to its lesser amount of sample used. In contrast to icELISA-I, icELISA-II is more convenient, whereas it consumes more reagents of coating antigen and goat anti-mouse IgG-peroxidase.
Increasing evidence shows that Epstein-Barr virus (EBV) infection is closely related to various lymphoid and epithelioid malignancies. However, the underlying mechanisms are unclear. GCNT3 (core 2β-1,6-acetylglucosaminyltransferase) is a new type of core mucin synthase, and its expression in EBV-associated gastric cancer (EBVaGC) is lower than that in EBV-negative gastric cancer (EBVnGC). EBV-encoded latent membrane protein 2A (LMP2A) is a transmembrane protein with tumorigenic transformation properties. Here, we demonstrated that LMP2A inhibited the transcription of GCNT3 by inhibiting Smad2/3 and Smad4. LMP2A restrained the activation of the mTORC1 pathway by inactivating the TGF-β1/Smad pathway and then downregulated GCNT3 expression. The mTORC1-GCNT3 pathway promoted cell proliferation and migration and inhibited G0/G1 cell arrest. Related proteins involved in epithelial-mesenchymal transition (EMT) were downstream molecules of the TGF-β1/Smad-mTORC1-GCNT3 pathway. GCNT3 inhibited autophagy by inducing mTORC1 phosphorylation. These findings indicate that targeting the TGF-β1/Smad-mTORC1-GCNT3 axis may represent a novel therapeutic target in GC.
Importance
Epstein-Barr virus (EBV) is an opportunistic pathogen, and the latent membrane protein 2A (LMP2A) encoded by EBV plays a key role in ensuring the incubation period of EBV. Glycosylation modification is an important marker of cancer cells, and recent studies have reported that it is related to EBV. Our conclusions provide deeper theoretical support for the role of LMP2A and TGF/Smad-mTORC1-GCNT3 in EBVaGC and help to understand glycosylation abnormalities in cancer. Our results may provide novel therapeutic targets for the treatment of gastric cancer against the TGF/Smad-mTORC1-GCNT3 signaling cascade.
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