Caffeine Abolishes the Ultraviolet-Induced REV3 Translesion Replication Pathway in Mouse Cells

When a replicative DNA polymerase stalls upon encountering a photoproduct on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion. Using an alkaline sucrose density gradient sedimentation technique, we previously classified this process termed UV-induced translesion replication (UV-TLS) into two types. In human cancer cells or xeroderma pigmentosum variant (XP-V) cells, UV-TLS was inhibited by caffeine or proteasome inhibitors. However, in normal human cells, the process was insensitive to these reagents. Reportedly, in yeast or mammalian cells, REV3 protein (a catalytic subunit of DNA polymerase ζ) is predominantly involved in the former type of TLS. Here, we studied UV-TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). In the wild-type MEF, UV-TLS was slow (similar to that of human cancer cells or XP-V cells), and was abolished by caffeine or MG-262. In 2 cell lines of Rev3KO-MEF (Rev3−/− p53−/−), UV-TLS was not observed. In p53KO-MEF, which is a strict control for Rev3KO-MEF, the UV-TLS response was similar to that of the wild-type. Introduction of the Rev3 expression plasmid into Rev3KO-MEF restored the UV-TLS response in selected stable transformants. In some transformants, viability to UV was the same as that in the wild-type, and the death rate was increased by caffeine. Our findings indicate that REV3 is predominantly involved in UV-TLS in mouse cells, and that the REV3 translesion pathway is suppressed by caffeine or proteasome inhibitors

Qualitative Measurement of Pain by Analysing the Salivary Alpha Amylase

The purpose of the study was to develop a qualitative measurement for pain by measuring salivary alpha amylase (sAA). In this study, sAA levels were measured in order to investigate the relativity between fluctuation of sAA and pain on Institute of Cancer Research (ICR) mice by injection needle as a physical stimulus. Mice were chosen as an object of experiment since mice and human have similarities in both physical behaviour and biological behaviour. Here, ICR mice were divided into two groups; injected by Nanopass 33 (TERUMO Corporation, 100 μm and 200 μm in inner and outer diameter) and a control (without any injection). Whole saliva was collected from the oral cavity by micropipette for both groups. As a result, there was a significant difference in sAA levels between the two groups as the p value (probability value) was below than 0.01 (p < 0.01). Therefore, this is the first study showing the non-invasive method of qualitative pain measurement for minor damage caused by physical stimulus. The results demonstrate the potential of sAA as an indirect marker for pain and suggest that the present experimental situation is a suitable experimental model for measuring pain

Abnormal gait, reduced locomotor activity and impaired motor coordination in Dgcr2-deficient mice

AbstractIt has been suggested that the DGCR2 gene plays a role in the pathogenesis of 22q11.2 deletion syndrome. To analyze its function, we used our Dgcr2-knock-out/EGFP-knock-in mice (Dgcr2-KO mice). At 20-26 weeks of age, approximately 20% of Dgcr2-KO mice showed gait abnormalities with trembling and difficulty in balancing. Footprint test revealed awkward movements in Dgcr2-KO mice soon after they were placed on the floor. Once they started walking, their stride lengths were not different from wild-type mice. In short-term open field test, Dgcr2-KO mice travelled a significantly shorter distance and walked more slowly than wild-type mice during the initial 5min after being placed in a new environment. In long-term open field test, Dgcr2-KO mice exhibited reduced cage activity compared to wild-type mice on the first day, but not on later days. Dgcr2-KO mice showed reduced latency to fall in the rotarod test, and the latency was not improved in the 3-day test. Histology revealed sparseness of cerebellar Purkinje cells in Dgcr2-KO mice. Our results suggest that Dgcr2 plays a role in motor control related to Purkinje cell function and that the deficiency of DGCR2 contributes at least to some of the symptoms of patients of 22q11.2 deletion syndrome