Molecular Evolution and Functional Characterization of a Bifunctional Decarboxylase Involved in Lycopodium Alkaloid Biosynthesis.

Lycopodium alkaloids (LAs) are derived from lysine (Lys) and are found mainly in Huperziaceae and Lycopodiaceae. LAs are potentially useful against Alzheimer's disease, schizophrenia, and myasthenia gravis. Here, we cloned the bifunctional lysine/ornithine decarboxylase (L/ODC), the first gene involved in LA biosynthesis, from the LA-producing plants Lycopodium clavatum and Huperzia serrata We describe the in vitro and in vivo functional characterization of the L. clavatum L/ODC (LcL/ODC). The recombinant LcL/ODC preferentially catalyzed the decarboxylation of l-Lys over l-ornithine (l-Orn) by about 5 times. Transient expression of LcL/ODC fused with the amino or carboxyl terminus of green fluorescent protein, in onion (Allium cepa) epidermal cells and Nicotiana benthamiana leaves, showed LcL/ODC localization in the cytosol. Transgenic tobacco (Nicotiana tabacum) hairy roots and Arabidopsis (Arabidopsis thaliana) plants expressing LcL/ODC enhanced the production of a Lys-derived alkaloid, anabasine, and cadaverine, respectively, thus, confirming the function of LcL/ODC in plants. In addition, we present an example of the convergent evolution of plant Lys decarboxylase that resulted in the production of Lys-derived alkaloids in Leguminosae (legumes) and Lycopodiaceae (clubmosses). This convergent evolution event probably occurred via the promiscuous functions of the ancestral Orn decarboxylase, which is an enzyme involved in the primary metabolism of polyamine. The positive selection sites were detected by statistical analyses using phylogenetic trees and were confirmed by site-directed mutagenesis, suggesting the importance of those sites in granting the promiscuous function to Lys decarboxylase while retaining the ancestral Orn decarboxylase function. This study contributes to a better understanding of LA biosynthesis and the molecular evolution of plant Lys decarboxylase.

Factors supporting long-term culture of bovine male germ cells.

Spermatogonial stem cells (SSCs) are unipotent in nature, but mouse SSCs acquire pluripotency under the appropriate culture conditions. Although culture systems are available for rodent and human germ-cell lines, no proven culture system is yet available for livestock species. Here, we examined growth factors, matrix substrates and serum-free supplements to develop a defined system for culturing primitive germ cells (gonocytes) from neonatal bovine testis. Poly-L-lysine was a suitable substrate for selective inhibition of the growth of somatic cells and made it possible to maintain a higher gonocyte:somatic cell ratio than those maintained with gelatin, collagen or Dolichos biflorus agglutinin (DBA) substrates. Among the serum-free supplements tested in our culture medium, knockout serum replacement (KSR) supported the proliferation and survival of gonocytes better than the supplements B-27 and StemPro-SFM after sequential passages of colonies. Under our optimised culture conditions consisting of 15% KSR supplement on poly-L-lysine-coated dishes, the stem-cell and germ-cell potentials of the cultured gonocytes were maintained with normal karyotype for more than 2 months (over 13 passages). The proposed culture system, which can maintain a population of proliferating bovine germ stem cells, could be useful for studying SSC biology and germline modifications in livestock animals.

The role of signaling pathways on proliferation and self-renewal of cultured bovine primitive germ cells.

Purpose: Gonocytes are primitive male germ cells residing in the neonatal testes and are unipotent in nature, but also have pluripotent stem cell ability in mice under appropriate culture conditions. This study was performed to elucidate the molecular mechanisms of self-renewal and survival of cultured bovine gonocytes. Methods: Gonocytes were isolated from neonatal bull calves and were cultured in DMEM/F12 supplemented with 15 % knock-out serum replacement (KSR) and glial cell-derived neurotrophic factor (GDNF). Cells were analyzed six days after culturing for cell-signaling molecular markers. Results: Colony formation was observed 3-4 days after being cultured. Addition of GDNF enhanced mitogen-activated protein kinase 1/2 (MAPK1/2) phosphorylation and activated the MAPK signaling pathway. Inhibition of MAPK signaling reduced cell proliferation and abolished colony formation. However, inhibition of phosphoinositide 3-kinase-AKT (PI3K-AKT) signaling, a dominant pathway for self-renewal of mouse germ cells, did not show any effects on cultured bovine gonocytes. Expression of cell cycle-related regulators cyclin D2 and cyclin-dependent kinase 2 (CDK2) was downregulated with inhibition of MAPK signaling. Conclusions: These results indicate activation of MAPK plays a critical role in self-renewal and survival of bovine gonocytes via cyclin D1 and CDK2.

Cranial molding helmet therapy and establishment of practical criteria for management in Asian infant positional head deformity.

BACKGROUND: The growing number of infants with deformational plagiocephaly (DP) has raised clinical questions about which children, at what age, and how molding helmet therapy (MHT) should be performed especially in Japan. METHODS: A total of 1,011 Japanese pediatric head deformity infants had undergone MHT after being diagnosed with non-synostotic DP. Three ratios of left to right comparison (anterior, posterior, and overall) were created and analyzed comparing age of starting treatment, helmet wearing period, and severity of skull deformity before with after MHT. RESULTS: The averages of head symmetry ratios after treatment in all groups (for the occipital region) showed apparent improvement; t(930) = -60.86, p = 0.000. (t(932) = -57.8, p = 0.000.) In the "severe" deformation group, the earlier the treatment was started, the higher symmetry ratio recovery was obtained. Treatment was especially effective when started in 4-month-old infants. In contrast to the "severe" group, the "mild" deformation group showed that MHT was most effective if treatment started before 6 months of age. Again, the earlier the treatment was started, the higher symmetry ratio was achieved, but compared to the "severe" group, it had a modest effect when treatment was started in infants older than 8 months. CONCLUSION: This is the first large-scale molding helmet study reporting the method and efficacy in Japanese infants. It demonstrated that despite the structural and physiological differences from infants of other races, molding helmet therapy is effective in Asian-born infants, provided that intervention timing and recognition conditions are met.

Collaborative Study of Thresholds for Mutagens: Adaptive Responses in the Micronucleus Test and Gene Induction by Mutagenic Treatments.

Background: We have been conducting a collaborative study on the thresholds of mutagens. In our previous examinations of cell activity and cell proliferation as endpoints, both displayed hormesis. This time, we conducted experiments to determine thresholds using the micronucleus test as an endpoint. Methods: The micronucleus test was conducted using Chinese hamster CHL/IU cells and mouse lymphoid L5178Y cells. Additionally, we conducted preliminary investigations into the gene expression using human TK6 cells. Results: When adhesive CHL/IU cells were treated with mitomycin C (MMC), and the hormetic response was examined, hormesis was not observed clearly. When L5178Y cells were treated with methyl methanesulfonate (EMS), AF-2, MMC, and colchicine, all of them exhibited an adaptive response. Additionally, cross-adaptive responses using AF-2 and MMC or EMS and MMC were conducted, both combinations showed a cross-adaptive response. When the gene expression patterns of six genes were investigated by RT-PCR after treatment with MMC, EMS, and H2O2 using TK6 cells, two genes, GADD45 A and P21, were induced in a dose- and time-dependent manner. Conclusion: Adaptive responses arise from preconditioning. As hormesis is inherently linked to preconditioning, adaptive responses observed in this study strongly suggest that hormesis was induced, hence existence of thresholds.

Multisite studies for optimization of a highly efficient culture assay used for in vitro detection of residual undifferentiated human pluripotent stem cells intermingled in cell therapy products.

Introduction: MEASURE2 (Multisite Evaluation Study on Analytical Methods for Non-clinical Safety Assessment of HUman-derived REgenerative Medical Products 2) is a Japanese experimental public-private partnership initiative that aims to standardize testing methods for tumorigenicity evaluation of human pluripotent stem cell (hPSC)-derived cell therapy products (CTPs). MEASURE2 organized multisite studies to optimize the methodology of the highly efficient culture (HEC) assay, a sensitive culture-based in vitro assay for detecting residual undifferentiated hPSCs in CTPs. Methods: In these multisite studies, 1) the efficiency of colony formation by human induced pluripotent stem cells (hiPSCs) under two different culture conditions and 2) the sorting efficiency of microbeads conjugated to various anti-hPSC markers during hiPSC enrichment were evaluated using samples in which hiPSCs were spiked into hiPSC-derived mesenchymal stem cells. Results: The efficiency of colony formation was significantly higher under culture conditions with the combination of Chroman 1, Emricasan, Polyamines, and Trans-ISRIB (CEPT) than with Y-27632, which is widely used for the survival of hPSCs. Between-laboratory variance was also smaller under the condition with CEPT than with Y-27632. The sorting efficiency of microbeads conjugated with the anti-Tra-1-60 antibody was sufficiently higher (>80%) than those of the other various microbeads investigated. Conclusions: Results of these multisite studies are expected to contribute to improvements in the sensitivity and robustness of the HEC assay, as well as to the future standardization of the tumorigenicity risk assessment of hPSC-derived CTPs.

Collaborative Study of Thresholds for Mutagens: Hormetic Responses in Cell Proliferation Tests Using Human and Murine Lymphoid Cells.

BACKGROUND: We previously showed that hormetic responses can be established in cell activity tests using human and murine adherent cells. This time, we examined whether hormetic responses can be established in cell proliferation tests using suspended human and murine lymphoid cells. METHODS: Human lymphoblastoid cells (TK6) and mouse lymphoma cells (L5178Y) were cultured in multi-well culture plates and treated with mitomycin C, ethyl methansulfonate, hygromycin B, aclarubicin or colchicine at various dose levels and the number of cells was measured at varied times using a flow cytometer. RESULTS: When the ratio of the number of cells treated with a test chemical to those in the negative control was plotted, the dose-response relationship typically showed a reverse U-shaped curve, indicating the occurrence of hormesis and existence of thresholds in cell toxicity. The hormetic responses depended largely on the test chemical, dose level and exposure time. When examining responses over the course of time, a J-shaped or fallen S-shaped curve was also observed. CONCLUSIONS: The dose-response relationship showed a reverse U-shaped curve, a hallmark of hormesis, at least some time points for all chemicals tested here, indicating that chemical hormesis can be established in in vitro cell proliferation tests.

Comprehensive evaluation of antioxidant effects of Japanese Kampo medicines led to identification of Tsudosan formulation as a potent antioxidant agent.

Oxidative stress due to the overproduction of reactive oxygen species plays an important role in the pathogenesis of various diseases. In the present study, we comprehensively evaluated the antioxidant activities of 147 oral formulations of Japanese traditional herbal medicines (Kampo medicines), representing the entire panel of oral Kampo medicines listed in the Japanese National Health Insurance Drug List, using in vitro radical scavenging assays, including the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity assay, the superoxide anion scavenging activity assay, and the oxygen radical absorption capacity assay. Three of the formulations tested, namely, Tsudosan, Daisaikoto, and Masiningan, showed the most potent in vitro antioxidant activities and were selected for further investigation of their intracellular and in vivo antioxidant effects. The results of the 2',7'-dichlorodihydrofluorescin diacetate assay demonstrated that all three Kampo medicines significantly inhibited hydrogen peroxide-induced oxidative stress in human hepatocellular liver carcinoma HepG2 cells. In addition, Tsudosan significantly increased the serum biological antioxidant potential values when orally administrated to mice, indicating that it also had in vivo antioxidant activity. The potent antioxidant activity of Tsudosan may be one of the mechanisms closely correlated to its clinical usage against blood stasis.

Collaborative study of thresholds for mutagens: proposal of a typical protocol for detection of hormetic responses in cytotoxicity tests.

BACKGROUND: According to the linear no-threshold model (LNT), even the smallest amount of radiation is hazardous. Although the LNT is not based on solid data, this hypothesis has been applied to mutagens and carcinogens. As a result, it has been postulated that there are no thresholds for these chemicals. To demonstrate negativity by experiments is practically impossible, because negative data may leave behind the possibility that additional data might make the resolution power high enough to change negativity to positivity. Furthermore, additional data collection may be endless and we may be trapped in agnosticism. When hormesis is established, in which biological responses are higher at low-doses and lower at high-doses than the control, thresholds could be established between the low- and high-doses. Before examination of thresholds in chemical mutagenesis, hormetic responses in cytotoxicity were tested using cultured mammalian cells. METHOD: Human cells (HeLa S3 and TK6) or Chinese hamster cells (CHL/IU) were cultured in 96-well plates and treated with mitomycin C (MMC) or ethyl methanesulfonate (EMS) at various dose levels and optical density was measured after addition of a reagent to detect cellular activity. In hormetic responses, data might fluctuate to and fro; therefore, experimental conditions were examined from various aspects to eliminate confounding factors including cell numbers, detection time, the edge effect of 96-well plates, and measurement time after addition of the reagent for detection. RESULTS: The dose response relationship was never linear. Cellular activities after treatment with MMC or EMS were generally higher at lower doses levels and lower at higher doses than the control, showing hormesis and allowing the establishment of thresholds. Dose response curves sometimes showed two or three peaks, probably reflecting different cellular responses. CONCLUSION: Hormetic responses in cytotoxicity tests were observed and thresholds could be established. Based on the results of this investigation, we put forward a tentative protocol to detect chemical hormesis in cytotoxicity tests, i.e., inoculate 2000 cells per well, add various doses of a test chemical 48 h after inoculation, add a detection dye 10 h after treatment, and measure optical density 2 h after addition of the reagent for detection.

Placental alkaline phosphatase levels in cerebrospinal fluid can have a decisive role in the differential diagnosis of intracranial germ cell tumors.

OBJECTIVE: Placental alkaline phosphatase (PLAP) in CSF can provide a very high diagnostic value in cases of intracranial germ cell tumors (GCTs), especially in pure germinomas, to the level of not requiring histological confirmation. Unlike other tumor markers, reliable data analysis with respect to the diagnostic value of PLAP serum or CSF levels has not been available until now. This is the first systematic and comprehensive study examining the diagnostic value of CSF PLAP in patients with intracranial GCTs. METHODS: From 2004 to 2014, 74 patients (average age 19.6 ± 10.6 years) with intracranial GCTs were evaluated using PLAP from their CSF and histological samples. Chemiluminescent enzyme immunoassay was utilized to measure CSF PLAP in the following tumor sites: pineal (n = 32), pituitary stalk, suprasellar (n = 16), basal ganglia (n = 15), intraventricular (n = 9), and cerebellar (n = 5) regions. In addition to classifying GCT cases, all patients underwent tumor biopsy for correlation with tumor marker data. RESULTS: PLAP in combination with other tumor markers resulted in extremely high sensitivity and specificity of the diagnostic value of intracranial GCTs. Intracranial GCT cases were classified into 1) germinomas, both "pure" and syncytiotrophoblastic giant cell types (n = 38); 2) nongerminomatous GCTs, choriocarcinomas (n = 9) and teratomas (n = 4); and 3) nongerminomas, other kinds of tumors (n = 23). Consequently, all patients received chemoradiation therapy based on elevation of PLAP and the histopathological results. It was also speculated that the level of PLAP could show the amount of intracranial germ cell components of a GCT. PLAP was 100% upregulated in all intracranial germinoma cases. The absence of CSF PLAP proved that the tumor was not a germinoma. CONCLUSIONS: The current study is the first systematic and comprehensive examination of the diagnostic value of the tumor marker PLAP in pediatric patients with intracranial GCT. Using the level of PLAP in CSF, we were able to detect the instances of intracranial germinoma with very high reliability, equivalent to a pathological diagnosis.

Genotoxicity studies OF p-dimethylaminoazobenzene (DAB).

The potential genotoxicity of the rodent liver carcinogen p-dimethylaminoazobenzene (DAB) was evaluated in compliance with the guidelines for genotoxicity studies of drugs (Notification No. 1604, Nov. 1, 1999, Ministry of Health and Welfare, Japan) and the OECD guidelines for testing chemicals. DAB was clearly positive in both the bacterial reverse mutation test (Ames test) and in vitro chromosomal aberration test in the presence of metabolic activation, whereas it was weakly positive at toxic doses in the rat bone marrow micronucleus test. It has been reported that DAB was clearly positive in in vivo genotoxicity tests, i.e., a mouse alkaline single cell gel electrophoresis (comet) assay and a young rat liver micronucleus test. These results suggest that the test system using the liver is effective for in vivo genotoxicity assessment of chemicals that show mutagenicity in in vitro genotoxicity tests in the presence of metabolic activation.