PD-L2, a newly identified member of B7 family, plays a role in down-regulating T cell responses. The common PD-L2 mRNA (type I) is the splicing product containing all 6 exons. We report here the identification of two human PD-L2 splice variants in activated leukocytes. One splice variant (type II) is generated through splicing out exon 3 encoding Ig constant-like domain; it retains all other regions without a frame shift. The other variant (type III) is created by splicing out exon 3 to an alternative acceptor site 5 bp downstream of the canonical acceptor site, leading to a frame shift. Consequently, the translated protein should be a soluble form. Furthermore, type I isoform is expressed on the plasma surface whereas type II isoform showed a pattern of intracellular membrane distribution in the transiently transfected K562 cells. In addition, the expression patterns of PD-L2 splice variants are variable in different individuals and distinct cellular status. These results suggest that PD-L2 expression may be controlled by posttranscriptional regulation through alternative splicing, and modulation of PD-L2 isoform expression may influence the outcome of immune response.
Indoleamine 2,3-dioxygenase (IDO) has been implicated in preventing the fetus from undergoing maternal T cell-mediated immune responses, yet the mechanism underlying these kinds of IDO-mediated immune responses has not been fully elucidated. Since the CD4 molecule plays a central role in the onset and regulation of antigen-specific immune responses, and T cell is sensitive in the absence of tryptophan, we hypothesize that IDO may reduce cell surface CD4 expression. To test this hypothesis, an adenoviral vector-based construct IDO-EGFP was generated and the effect of IDO-EGFP on CD4 expression was determined on recombinant adenoviral infected C8166 and MT-2 cells, by flow cytometry and/or Western blot analysis. The results revealed a significant downregulation of cell membrane CD4 in pAd-IDOEGFP infected cells when compared to that of mock-infected cells or infection with empty vector pAd-EGFP. Further experiments disclosed that either an addition of tryptophan or IDO inhibitor could partly restore CD4 expression in pAd-IDOEGFP infected C8166 cells. Our findings suggest that downregulation of CD4 by IDO might be one of the mechanisms through which IDO regulates T cell-mediated immune responses.
Small ubiquitin-related modifier (SUMO) fusion system has been shown to be efficient for enhancing expression and preventing degradation of the target protein. We showed herein that SUMO fusion to human keratinocyte growth factor 2 (hKGF-2) gene was feasible and it significantly enhanced protein expression and its efficiency. The fusion DNA fragment composed of SUMO gene, which was fused to hexahistidine tag, and hKGF-2 gene was amplified by PCR and inserted into the expression vector pET28a to construct the recombinant plasmid, pET28a-SUMO-hKGF-2. The plasmid was then transformed into Escherichia coli Rosetta(TM)2(DE3), and the recombinant fusion protein SUMO-hKGF-2 was expressed at 30 degrees C for 6 h, with the induction of IPTG at the final concentration of 0.4 mM. The expression level of the fusion protein was up to 30% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography. After desalting by Sephadex G-25 size exclusion chromatography, the hexahistidine-SUMO-hKGF-2 was digested by SUMO proteases. The recombinant hKGF-2 was purified again with Ni-NTA column and the purity was about 95% with a total yield of 13.9 mg/l culture. The result of mitogenicity assay suggests that the recombinant hKGF-2 can significantly promote the proliferation of normal rat kidney epithelial (NRK-52E) cells.
Bacillus velezensis zk1 is the dominant bacterium that causing rot in peaches. However, the mechanisms through that this bacterium causes rot have not been elucidated. Here, we explored the mechanisms of peach decay caused by B. velezensis zk1. The invasion of B. velezensis zk1 in peaches resulted in an increase in glucose and arabinose contents in fruit tissues. Moreover, the relative conductivity of the fruit reached 84% after 4 days of culture with bacterial invasion. With the destruction of cells, the malondialdehyde content increased, whereas the vitamin C, dialdehyde, flavonoid and total phenol contents decreased. Polyphenol oxidase, superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and ammonia lyase activities also decreased. Overall, these findings demonstrated that B. velezensis zk1 infection damaged peach chloroplasts, mitochondria, respiratory chain activity and related free radical scavenging enzyme systems, thereby disrupting the normal physiological metabolism of peaches and causing rot.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.