Calcineurin knockout mice show the inhibition of motor functionsloss of synaptic plasticitylearning and memory

ATRIP has several functions in ATR signaling including stabilizing the ATR protein, targeting ATR to replication stress sites, and contributing to the interaction with the TOPBP1 protein. TOPBP1 binding to the ATR-ATRIP complex activates ATR by inducing an unknown structural change within ATR that increases ATR substrate affinity. The mutations creating a hyperactive kinase may partly mimic the effect of TOPBP1 binding to ATR-ATRIP and potentiate the ability of TOPBP1 to promote the change in ATR conformation needed for its increased activity. In summary, we identified single amino acid mutations within the ATR HEAT repeats that alter its kinase activity. Cells expressing S1333A-ATR have elevated basal phosphorylation levels of ATR substrates but no noticeable checkpoint or replication defects in cultured cells. Thus, cells can tolerate elevated basal ATR kinase activity. The small decrease in ATR activity caused by the S1333D mutation is enough to cause modest defects in some ATR checkpoint functions. S1333 is not in a region of ATR previously known to be involved in regulation of the kinase. Future high-resolution structural studies will aid in understanding why this region is important to regulate ATR activity levels. The accumulation of green fluorescent protein in cells is widely used as a molecular tag that can be readily visualized under ultraviolet light illumination. Many different GFP-transgenic animals have been generated and utilized for tracking cells in organ and cell transplantation studies. GFP can show weak immunogenicity and/or cell toxicity that can potentially alter experimental results. Gambotto et al. showed that GFP could generate an antigenic epitope that binds to H2-Kd molecules in BALB/c mice, while Inoue and colleagues generated the GFP-Tg Lewis rat and reported that transplanted skin grafts from these rats to wild-type Lewis rats lost viability after about a week, suggesting immunological rejection. Nevertheless, isolated cells from GFP-Tg rats were observed long after cell transplantation into immune-privileged sites such as the central nervous system and joints. In addition, liver harvested from a GFP-Tg Lewis rat survived long term in a wild-type Lewis rat without the use of an immunosuppressant. These experimental findings imply that GFP is weakly immunogenic, but that organs or cells expressing GFP can survive at sites where there is a weaker immunological reaction. In general, transplanted allogeneic hepatocytes are eliminated within a few days without the use of an immunosuppressant. Nevertheless, studies with rat models suggest that GFP is minimally immunogenic when GFP-positive hepatocytes or stem/progenitor cells are transplanted into syngeneic liver. Oertel and colleagues transplanted hepatocytes transfected with the GFP gene into retrorsine-pretreated wild-type syngeneic rat liver. They demonstrated continuous GFP expression, driven by the liver-specific albumin enhancer/promoter, in transplanted hepatocytes up to four months after transplantation. Other studies showed repopulation of injured liver tissue by transplanted syngeneic stem/progenitor cells expressing GFP. Therefore, we expected to see long-term survival of GFP-positive hepatocytes after transplantation into a wild-type Lewis rat liver. In a pilot study, we did not observe proliferation of GFP-positive hepatocytes at six weeks after transplantation of a syngeneic liver specimen. This observation was considered to be important not only for the interpretation of previous data, but also in planning of future experiments using the rat model containing GFP-positive hepatocytes. Therefore, further studies were performed to answer three questions. 1) Did a technical error occur that prevented proliferation of GFP-positive hepatocytes? 2) Was there a loss of GFP-positive hepatocytes or a loss of GFP expression? 3) Was this phenomenon caused by a host immunological response or by GFP toxicity? This study demonstrated that GFP-positive hepatocytes isolated from GFP-Tg rats could engraft in wild-type host rats. Importantly, the transplanted cells did not persist for more than 42 days in a wild-type syngeneic rat liver that was pretreated with retrorsine and by partial hepatectomy. In contrast, hepatocytes transplanted from wild-type rats steadily proliferated in GFP-Tg Lewis rat liver. Immunosuppressant treatment with tacrolimus prolonged the survival of GFP-positive hepatocytes, whereas preimmunization with GFP-Tg hepatocytes decreased the time to disappearance of transplanted hepatocytes in wild-type rats. Prolonged survival of GFP-positive hepatocytes by bone marrow transplantation eliminated the potential protective effect of tacrolimus on GFP-Tg hepatocytes. These results strongly suggest that the disappearance of transplanted hepatocytes in our model was primarily due to an immunological reaction to the GFP transgene rather than to GFP toxicity. GFP-Tg Lewis rats were originally generated using Lewis rats obtained from Charles-River Laboratories Japan, the rats exported from Charles-River Laboratories in the USA in 1981. We initially noticed the disappearance of transplanted hepatocytes by using wild-type Lewis rats from Harlan Sprague- Dawley, and hypothesized that these two rats from two different colonies might express different antigens affecting the immunological reaction. In fact, the phenomenon was reproduced in wildtype Lewis rats from Charles-River Laboratories. Therefore, we consider that the cellular loss after GFP-positive hepatocyte transplantation is due to an immunological reaction against GFP.