cDNA clones containing partial sequences for beta-glucuronidase (beta G) were constructed from rat preputial gland RNA and identified by their ability to selectively hybridize beta G mRNA. One such rat clone was used to isolate several cross-hybridizing clones from a mouse-cDNA library prepared from kidney RNA from androgen-treated animals. Together, the set of mouse clones spans about 2.0 kb of the 2.6-kb beta G mRNA. Using these cDNA clones as probes, a genomic polymorphism for DNA restriction fragment size was found that proved to be genetically linked to the beta G gene complex. A fragment of beta G cDNA was subcloned into a vector carrying an SP6 polymerase promoter to provide a template for the in vitro synthesis of single-stranded RNA complementary to beta G mRNA. This provided an extremely sensitive probe for the assay of beta G mRNA sequences. Using either nick-translated cDNA or transcribed RNA as a hybridization probe, we found that mouse beta G RNA levels are strongly induced by testosterone, and that induction by testosterone is pituitary-dependent. During the lag period preceding induction, during the induction period itself, and during deinduction following removal of testosterone, beta G mRNA levels paralleled rates of beta G synthesis previously measured by in vivo pulse-labelling experiments. Genetic variation in the extent of induction affected either the level of beta G mRNA or its efficiency of translation depending on the strain of mice tested.
Rejection is still a critical barrier to the long-term survival of graft after liver transplantation, requiring clinicians to unveil the underlying mechanism of liver transplant rejection. The cellular diversity and the interplay between immune cells in the liver graft microenvironment remain unclear. Herein, we performed single-cell RNA sequencing analysis to delineate the landscape of immune cells heterogeneity in liver transplantation. T cells, NK cells, B cells, and myeloid cell subsets in human liver and blood were enriched to characterize their tissue distribution, gene expression, and functional modules. The proportion of CCR6+CD4+ T cells increased within an allograft, suggesting that there are more memory CD4+ T cells after transplantation, in parallel with exhausted CTLA4+CD8+ T and actively proliferating MKI67+CD8+ T cells increased significantly, where they manifested heterogeneity, distinct function, and homeostatic proliferation. Remarkably, the changes of CD1c+ DC, CADM+ DC, MDSC, and FOLR3+ Kupffer cells increase significantly, but the proportion of CD163+ Kupffer, APOE+ Kupffer, and GZMA+ Kupffer decreased. Furthermore, we identified LDLR as a novel marker of activated MDSC to prevent liver transplant rejection. Intriguingly, a subset of CD4+CD8+FOXP3+ T cells included in CTLA4+CD8+ T cells was first detected in human liver transplantation. Furthermore, intercellular communication and gene regulatory analysis implicated the LDLR+ MDSC and CTLA4+CD8+ T cells interact through TIGIT-NECTIN2 signaling pathway. Taken together, these findings have gained novel mechanistic insights for understanding the immune landscape in liver transplantation, and it outlines the characteristics of immune cells and provides potential therapeutic targets in liver transplant rejection.
We have developed a novel cell-based protein-protein interaction assay method. The method relies on conversion of an inactive permuted luciferase containing a Tobacco Etch Virus protease (TEV) cleavage sequence fused onto protein (A) to an active luciferase upon interaction and cleavage by another protein (B) fused with the TEV protease. We demonstrate assay applicability for ligand-induced protein-protein interactions including G-protein coupled receptors, receptor tyrosine kinases and nuclear hormone receptors.
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