A large intermediate domain of vertebrate REV3 protein is dispensable for ultraviolet-induced translesion replication.

DNA polymerase ζ (pol ζ) is involved in translesion replication (translesion synthesis, TLS) and plays an essential role in embryogenesis. In adults, pol ζ triggers mutation as a result of error-prone TLS and causes carcinogenesis. The catalytic subunit of pol ζ, REV3, is evolutionarily conserved from yeast and plants to higher eukaryotes. However, the structures are notably different: unlike that in yeast REV3, a large intermediate domain is inserted in REV3 of humans and mice. The domain is mostly occupied with noncommittal structures (random coil…etc.); therefore, its role and function are yet to be resolved. Previously, we reported deficient levels of ultraviolet (UV)-induced TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). Here, we constructed a mouse Rev3-expressing plasmid with a deleted intermediate domain (532-1793 a.a,) and transfected it into Rev3KO-MEF. The isolated stable transformants showed comparable levels of UV-sensitivity and UV-TLS activity to those in wild-type MEF, detected using an alkaline sucrose density gradient sedimentation. These results indicate that the intermediate domain is nonessential for UV-induced translesion replication in cultured mouse cells.

"Kampo-sommelier Practice": A Trial for an Active Learning Program in Kampo (Japanese Traditional) Medicine.

OBJECTIVE: This study aimed to assess the effectiveness of Kampo-sommelier practice, an active learning program on crude drugs used in Kampo formulations. METHODS: The participants were fourth-year Tokai University School of Medicine students as of 2017. Eighteen small teams attended a 20-minute Kampo-sommelier practice session and were provided 10 kinds of crude drugs (Licorice, Cinnamon, Ginger, etc.) in three forms, original, cut, and powdered, while blinded to the drugs. Each team was asked to distinguish each drug in terms of form, scent, flavor, and color with reference to described characteristics. The ability to match the names of the drugs with their descriptions was assessed in the participants one month later, and also in human science "A" and medicine "B" students, without prior education, and pharmacy "C" students, with professional education. RESULTS: The 117 participants received an average score of 6.2 ± 2.4 (mean ± S.D.) out of 10, which was significantly higher than 3.4 ± 1.8 in 97 "A" students and 3.1 ± 2.4 in 85 "B" students and lower than 8.4 ± 2.1 in 135 "C" students (p < 0.05 for all). CONCLUSIONS: The effectiveness of this team-based learning approach is suggested by the significantly higher scores of the participants.

A mutation in NOTCH1 ligand binding region detected in patients with oral squamous cell carcinoma reduces NOTCH1 oncogenic effect.

NOTCH1 is known as an oncogenic or tumor suppressive gene in solid cancer. NOTCH1 mutations in oral squamous cell carcinoma (OSCC) frequently occur near the ligand-binding region. These mutations change the domain structure of this protein and affect the ligand binding activity. When NOTCH1 is activated by ligand binding, NOTCH1 intracellular domain (NICD) is cleaved from the cell membrane. This study investigated the functional change induced by a NOTCH1 mutation detected in OSCC clinical samples using stable transformant analysis. HEK293 cell lines expressing NOTCH1 wild-type (WT cells) or p.A465T NOTCH1 (A465T cells) were established. NOTCH1 expression was analyzed by flow cytometry, western blotting, and immunofluorescence using an anti-human NOTCH1 antibody. mRNA expression levels in WT and A465T cells were determined by quantitative real-time PCR (qPCR). Cell proliferation was analyzed by using cell growth assays and a xenograft tumor assay. Flow cytometry indicated that NOTCH1 expression on the cell membrane was lower in A465T cells than that in WT cells. NOTCH1 and NICD were both detected by western blot in WT and A465T cells. The immunofluorescence signal for NICD was detected in the nucleus of WT cells, while it was localized mainly in the cytoplasm of A465T cells. HES1 and HEY1 mRNA expression levels were lower in A465T than in WT cells. The cell growth of WT cells was significantly higher than that of HEK293 cells (3-fold, P<0.01), while that of A465T cells was significantly lower than that of HEK293 cells (37%, P<0.01). In a xenograft model, the tumor cell implantation rate of WT cells was 80%, while that of A465T cells was 0%. This study indicates that NOTCH1 acts as an oncogene and that the NOTCH1 mutation (p.A465T) in the ligand-binding region causes the loss of tumorigenicity by downregulating the NOTCH1 pathway.

Microneedle pH Sensor: Direct, Label-Free, Real-Time Detection of Cerebrospinal Fluid and Bladder pH.

Acid-base homeostasis (body pH) inside the body is precisely controlled by the kidneys and lungs and buffer systems, such that even a minor pH change could severely affect many organs. Blood and urine pH tests are common in day-to-day clinical trials and require little effort for diagnosis. There is always a great demand for in vivo testing to understand more about body metabolism and to provide effective diagnosis and therapy. In this article, we report the simple fabrication of microneedle-based direct, label-free, and real-time pH sensors. The reference and working electrodes were Ag/AgCl thick films and ZnO thin films on tungsten (W) microneedles, respectively. The morphological and structural characteristics of microneedles were carefully investigated through various analytical methods. The developed sensor exhibited a Nernstian response of -46 mV/pH. Different conditions were used to test the sensor to confirm their accuracy and stability, such as various buffer solutions, with respect to time, and we compared the reading with commercial pH electrodes. Besides that, the fabricated microneedle sensor ability is proven by in vivo testing in mouse cerebrospinal fluid (CSF) and bladders. The pH sensor procedure reported here is totally reversible, and results were reproducible after several rounds of testing.

Preparation of Clinically Useful Sennoside-reduced Rhubarb.

OBJECTIVE: The aim of this study was to develop a method of removing sennoside to reduce the cathartic effect of rhubarb while conserving its other pharmacological activities. METHODS: Rhubarb powder was steam autoclaved at 121°C and 0.14 MPa for 20, 60, or 120 minutes, and HPLC analysis was conducted to determine levels of rhubarb components. Mice were fed non-autoclaved or 20-minute-autoclaved rhubarb extracts. Feces were collected and weighed over a 24-hour period. India ink was orally administered to determine the distance of fecal migration through the intestinal tract. RESULTS: Autoclaving 20, 60, and 120 minutes decreased sennoside A and B to trace levels but only autoclaving 20 minutes conserved most of the (+)-catechin, (-)-epicatechin, and (-)-epicatechin gallate contents (i.e., 69%, 90%, 88%, respectively). Therefore only rhubarb autoclaved for 20 minutes was used in subsequent experiments. Fecal output (in g) in mice treated with water (control), autoclaved rhubarb, and non-autoclaved rhubarb was 2.78 ± 0.07, 3.30 ± 0.13 (p = 0.348), and 3.81 ± 0.07 (p = 0.005). India ink migration was far less in mice treated with autoclaved rhubarb vs non-autoclaved rhubarb. CONCLUSION: Steam autoclaving the rhubarb for 20 minutes reduces sennoside levels and its cathartic activity while conserving its other pharmacological activities.

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

It 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 5 min 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.

Frequent mutations in NOTCH1 ligand-binding regions in Japanese oral squamous cell carcinoma.

Recent studies showed that head and neck squamous cell carcinoma (HNSCC) including oral squamous cell carcinoma (OSCC) of Caucasian, Chinese and Indian patients frequently have NOTCH1 mutations. We found eight of 84 OSCC in Japanese patients have point mutations (9.5%) correspond to the ligand binding region of NOTCH1 protein. Two set of them are the same mutations and all mutations are non-synonymous G>A transitions. In addition, median disease-free survival is significantly longer in patients with NOTCH1-mutated tumors as compared to those without the mutation (P<0.05). The protein structure simulation based on X-ray crystallography indicated that new p.A465T mutation leads to a conformational change of NOTCH1 ligand binding domain as well as the p.G481S mutant NOTCH1 with a loss of flexibility around this residue. These results suggest that NOTCH1 mutation occurs frequently in Japanese OSCC in the vicinity of the ligand binding region and, these mutations cause downregulation of the NOTCH1 function.

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.

Peony root extract upregulates transthyretin and phosphoglycerate mutase in mouse cobalt focus seizure.

Cobalt focus is a seizure focus model in which cerebral neurons exhibit long-lasting severe spike discharges, followed by neuronal death. However, the neuronal death is prevented when peony root extract (PR) is administered prior to cobalt application. We tested the hypothesis that PR modulates the expression of neuroprotective proteins in the cerebrum of mouse cobalt focus by proteomic analysis using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry to screen for differentially expressed proteins. Analyses revealed that transthyretin, a carrier protein for thyroid hormones and retinoids, and the brain form of phosphoglycerate mutase, a glycolytic enzyme, were upregulated in the cobalt-treated mouse cerebrum and further increased by PR administration in association with upregulation of neurogranin/RC3, a target of the transcriptional activation by thyroid hormones and retinoids. These findings suggest that PR-induced protection of mouse cerebral neurons involves neurotrophic events caused by thyroid hormones and/or retinoids and enhanced glycolysis.

Molecular mechanism of preventive effect of peony root extract on neuron damage.

The molecular mechanism of the protective effects of peony root extract and its component substances on neuron damage induced by the cobalt focus epilepsy model and the EL mouse was investigated. Long-term administration of peony root extract for 30 days prior to metallic cobalt powder application to the cerebral cortex of mice resulted in increased expression of A20, an inhibitor gene of cell death. In the EL mouse, a hereditary epilepsy animal model with vulnerable neurons, increased expression of A20 was observed even without administration of peony root extract. Long-term administration of peony root extract to the EL mouse resulted in a marked increase of expression of A20. These results suggested that an increase in A20 expression is the main molecular mechanism of protective action of peony root extract on neuron damage.

An essential role for REV3 in mammalian cell survival: absence of REV3 induces p53-independent embryonic death.

The REV3 gene of budding yeast encodes the catalytic subunit of DNA polymerase zeta that carries out translesion DNA synthesis. While REV3-null yeast mutants are viable and exhibit normal growth, Rev3-deficient mice die around midgestation of embryogenesis, which is accompanied by massive apoptosis of cells within the embryo proper. We have investigated whether REV3 is required for the survival of mouse cells and whether the embryonic lethality caused by REV3 deficiency can be rescued by introduction of a Rev3 transgene or by inactivation of p53, the cellular gatekeeper that regulates DNA damage-induced apoptosis. We show that Rev3(-/-) blastocysts were unable to survive and grow in culture but expression of a Rev3 transgene restored their outgrowth. Moreover, Rev3 transgene expression suppressed the apoptosis in E7.5 Rev3(-/-) embryos. The Rev3(-/-) embryonic lethality, however, was not rescued by either Rev3 transgene expression or p53 deficiency. These results reveal an essential role for REV3 in the survival and growth of mammalian cells and suggest that Rev3(-/-) embryonic death occurs in a p53-independent pathway.