• Journal of Microbiology and Biotechnology
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• 제17권7호
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• pp.1162-1168
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• 2007

Although widely used as a host for recombinant protein production, Escherichia coli is unsuitable for massive screening of recombinant clones, owing to its poor secretion of proteins. A vector system containing T4 holin and T7 lysozyme genes under the control of the ptsG promoter derivative that is inducible in the absence of glucose was developed for programmed cell lysis of E. coli. Because E. coli harboring the vector grows well in the presence of glucose, but is lysed upon glucose exhaustion, the activity of the foreign gene expressed in E. coli can be monitored easily without an additional step for cell disruption after the foreign gene is expressed sufficiently with an appropriate concentration of glucose. The effectiveness of the vector was demonstrated by efficient screening of the amylase gene from a Bacillus subtilis genomic library. This vector system is expected to provide a more efficient and economic screening of bioactive products from DNA libraries in large quantities.

• BMB Reports
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• 제31권2호
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• pp.183-188
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• 1998

2-chloroethylethyl sulfide (CEES) is an alkylating agent that readily reacts with a wide variety of biological molecules causing metabolic abnormality. The mechanism of cell death during CEES injury is poorly understood. We have examined the effect of exposure of thymocytes with various concentrations of CEES to determine the pattern of cell death in thymocytes injury induced by CEES. In the present study, we show that two patterns of cell death occurred by either one of two mechanisms: apoptosis and necrosis. Exposure to low level of CEES (100 ${\mu}M$) for 5 h caused an induction of apoptosis on thymocytes, as identified by the following criteria: DNA fragmentation visualized by the characteristic "ladder" pattern was observed upon agarose gel electrophoresis and morphological features were revealed by microscopical observations. In contrast, exposure to high levels of CEES (500 ${\mu}M$) induce necrotic features such as cell lysis. Thus, depending on the concentrations, CEES can result in either apoptotic or necrotic cell damage. Our findings suggest that thymocytes which are not killed directly, but merely injured by low levels of CEES, are able to activate an internally-programmed cell death mechanism, whereas thymocytes receiving severe damages apparently can not.

• Asian Pacific Journal of Cancer Prevention
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• 제13권8호
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• pp.4031-4036
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• 2012

Background: The negative signaling provided by interactions of the co-inhibitory molecule, programmed death-1 (PD-1), and its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), is a critical mechanism contributing to tumor evasion; blockade of this pathway has been proven to enhance cytotoxic activity and mediate antitumor therapy. Here we evaluated the anti-tumor efficacy of AAV-mediated delivery of the extracellular domain of murine PD-1 (sPD-1) to a tumor site. Material and Methods: An rAAV vector was constructed in which the expression of sPD-1, a known negative regulator of TCR signals, is driven by human cytomegalovirus immediate early promoter (CMV-P), using a triple plasmid transfection system. Tumor-bearing mice were then treated with the AAV/sPD1 construct and expression of sPD-1 in tumor tissues was determined by semi quantitative RT-PCR, and tumor weights and cytotoxic activity of splenocytes were measured. Results: Analysis of tumor homogenates revealed sPD-1 mRNA to be significantly overexpressed in rAAV/sPD-1 treated mice as compared with control levels. Its use for local gene therapy at the inoculation site of H22 hepatoma cells could inhibit tumor growth, also enhancing lysis of tumor cells by lymphocytes stimulated specifically with an antigen. In addition, PD-1 was also found expressed on the surfaces of activated CD8+ T cells. Conclusion: This study confirmed that expression of the soluble extracellular domain of PD-1 molecule could reduce tumor microenvironment inhibitory effects on T cells and enhance cytotoxicity. This suggests that it might be a potential target for development of therapies to augment T-cell responses in patients with malignancies.