RESUMEN
An improved method for the online preconcentration, derivatization, and separation of phosphorylated compounds was developed based on the affinity of a Phos-tag acrylamide gel formed at the intersection of a polydimethylsiloxane/glass multichannel microfluidic chip toward these compounds. The acrylamide solution comprised Phos-tag acrylamide, acrylamide, and N,N-methylene-bis-acrylamide, while 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] was used as a photocatalytic initiator. The Phos-tag acrylamide gel was formed around the channel crossing point via irradiation with a 365 nm LED laser. The phosphorylated peptides were specifically concentrated in the Phos-tag acrylamide gel by applying a voltage across the gel plug. After entrapment of the phosphorylated compounds in the Phos-tag acrylamide gel, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was introduced to the gel for online derivatization of the concentrated phosphorylated compounds. The online derivatized DTAF-labeled phosphorylated compounds were eluted by delivering a complex of phosphate ions and ethylenediamine tetraacetic acid as the separation buffer. This method enabled sensitive analysis of the phosphorylated peptides.
RESUMEN
Epstein-Barr virus (EBV), which infects not only B cells, but also T cells and natural killer (NK) cells, is associated with multiple lymphoid malignancies. Recently, the proteasome inhibitor bortezomib was reported to induce apoptosis of EBV-transformed B cells. We evaluated the killing effect of this proteasome inhibitor on EBV-associated T lymphoma cells and NK lymphoma cells. First, we found that bortezomib treatment decreased the viability of multiple T and NK cell lines. No significant difference was observed between EBV-positive and EBV-negative cell lines. The decreased viability in response to bortezomib treatment was abrogated by a pan-caspase inhibitor. The induction of apoptosis was confirmed by flow cytometric assessment of annexin V staining. Additionally, cleavage of caspases and polyadenosine diphosphate-ribose polymerase, increased expression of phosphorylated IκB, and decreased expression of inhibitor of apoptotic proteins were detected by immunoblotting in bortezomib-treated cell lines. We found that bortezomib induced lytic infection in EBV-positive T cell lines, although the existence of EBV did not modulate the killing effect of bortezomib. Finally, we administered bortezomib to peripheral blood mononuclear cells from five patients with EBV-associated lymphoproliferative diseases. Bortezomib had a greater killing effect on EBV-infected cells. These results indicate that bortezomib killed T or NK lymphoma cells by inducing apoptosis, regardless of the presence or absence of EBV.
