We investigated the role of the endoplasmic reticulum (ER) stress response in intracellular Ca 2؉ regulation, MAPK activation, and cytoprotection in LLC-PK 1 renal epithelial cells in an attempt to identify the mechanisms of protection afforded by ER stress. Cells preconditioned with trans-4,5-dihydroxy-1,2-dithiane, tunicamycin, thapsigargin, or A23187 expressed ER stress proteins and were resistant to subsequent H 2 O 2 -induced cell injury. In addition, ER stress preconditioning prevented the increase in intracellular Ca 2؉ concentration that normally follows H 2 O 2 exposure. Stable transfection of cells with antisense RNA targeted against GRP78 (pkASgrp78 cells) prevented GRP78 induction, disabled the ER stress response, sensitized cells to H 2 O 2 -induced injury, and prevented the development of tolerance to H 2 O 2 that normally occurs with preconditioning. ERK and JNK were transiently (30 -60 min) phosphorylated in response to H 2 O 2 . ER stress-preconditioned cells had more ERK and less JNK phosphorylation than control cells in response to H 2 O 2 exposure. Preincubation with a specific inhibitor of JNK activation or adenoviral infection with a construct that encodes constitutively active MEK1, the upstream activator of ERKs, also protected cells against H 2 O 2 toxicity. In contrast, the pkASgrp78 cells had less ERK and more JNK phosphorylation upon H 2 O 2 exposure. Expression of constitutively active ERK also conferred protection on native as well as pkASgrp78 cells. These results indicate that GRP78 plays an important role in the ER stress response and cytoprotection. ER stress preconditioning attenuates H 2 O 2 -induced cell injury in LLC-PK 1 cells by preventing an increase in intracellular Ca 2؉ concentration, potentiating ERK activation, and decreasing JNK activation. Thus, the ER stress response modulates the balance between ERK and JNK signaling pathways to prevent cell death after oxidative injury. Furthermore, ERK activation is an important downstream effector mechanism for cellular protection by ER stress.

Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell–virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71) 3C protease (3Cpro) cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3′ pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3Cpro. CstF-64 was cleaved in vitro by 3Cpro but neither by mutant 3Cpro (in which the catalytic site was inactivated) nor by another EV71 protease 2Apro. Serial mutagenesis was performed in CstF-64, revealing that the 3Cpro cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500). An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3′-end pre-mRNA processing and polyadenylation in 3Cpro-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3Cpro cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.

Intragastric inoculation of mice with Klebsiella pneumoniae can cause liver abscesses, necrosis of liver tissues, and bacteremia. A newly isolated phage (NK5) with lytic activity for K. pneumoniae was used to treat K. pneumoniae infection in an intragastric model. Both intraperitoneal and intragastric administration of a single dose of NK5 lower than 2 ؋ 10 8 PFU at 30 min after K. pneumoniae infection was able to protect mice from death in a dose-dependent manner, but the efficacy achieved with a low dose of NK5 by intragastric treatment provided the more significant protection. Phage NK5 administered as late as 24 h after K. pneumoniae inoculation was still protective, while intraperitoneal treatment with phage was more efficient than intragastric treatment as a result of the dissemination of bacteria into the circulation at 24 h postinfection. Surveys of bacterial counts for mice treated with NK5 by the intraperitoneal route revealed that the bacteria were eliminated effectively from both blood and liver tissue. K. pneumoniae-induced liver injury, such as liver necrosis, as well as blood levels of aspartate aminotransferase and alanine aminotransferase and inflammatory cytokine production, was significantly inhibited by NK5 treatment. These data suggest that a low dose of NK5 is a potential therapeutic agent for K. pneumoniae-induced liver infection.