Lignan and phenylpropanoid glycosides from Fernandoa adenophylla.

From the leaves and branches of Fernandoa adenophylla, a lignan glycoside (fernandoside) and a phenylpropanoid glycoside (2"-O-beta-apiosylverbascoside) were isolated together with 12 known compounds. The structural elucidations were based on analyses of physical and spectroscopic data.     

Identification of a highly immunogenic mouse breast cancer sub cell line, 4T1-S

Cancer vaccines serve as a promising clinical immunotherapeutic strategy that help to trigger an effective and specific antitumor immune response compared to conventional therapies. However, poor immunogenicity of tumor cells remains a major obstacle for clinical application, and developing new methods to modify the immunogenicity of tumor cells may help to improve the clinical outcome of cancer vaccines. 4T1 mouse breast cancer cell line has been known as poorly immunogenic and highly metastatic cell line. Using this model, we identified a sub cell line of 4T1—designated as 4T1-Sapporo (4T1-S)—which shows immunogenic properties when used as a vaccine against the same line. In 4T1-S-vaccinated mice, subcutaneous injection of 4T1-S resulted in an antitumor inflammatory response represented by significant enlargement of draining lymph nodes, accompanied with increased frequencies of activated CD8 T cells and a subpopulation of myeloid cells. Additionally, 4T1-S vaccine was ineffective to induce tumor rejection in nude mice, which importantly indicate that 4T1-S vaccine rely on T cell response to induce tumor rejection. Further analysis to identify mechanisms that control tumor immunogenicity in this model may help to develop new methods for improving the efficacies of clinical cancer vaccines.     

Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector

Replication-defective recombinant adenovirus, Adex4SRLacZL, was used as a vector for transferring exogenous genes in mouse zona pellucida-free eggs at the pronuclear stage. The vector contained the E. coli LacZ reporter gene under the control of the SRα promoter (SV40 early promoter-fused HTLV-I LTR), and the expression of the reporter gene was examined during preimplantation development in culture. Histochemical staining of the embryos for β-galactosidase activity showed that the exogenous LacZ gene as expressed in 98% of the embryos at the morula-blastocyst stages. As in the microinjection method, the exogenous genes could be pursued from the 2-cell stage. Neither apparent morphological changes nor cytotoxic effects were observed. Both the percentages of embryos expressing reporter genes and the rate of development to the blastocyst stage were higher in the adenovirus vector-treated embryos than in the microinjected ones. These results suggest that the adenovirus vector system is a useful tool in investigating the genetic control of early mammalian development. © 1995 wiley-Liss, Inc.     

JNK and p38 MAPK are independently involved in tributyltin-mediated cell death in rainbow trout (Oncorhynchus mykiss) RTG-2 cells

Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases that transmit various extracellular signals to the nucleus inducing gene expression, cell proliferation, and apoptosis. Recent studies have revealed that organotin compounds induce apoptosis and MAPK phosphorylation/activation in mammal cells. In this study, we elucidated the cytotoxic mechanism of tributyltin (TBT), a representative organotin compound, in rainbow trout (Oncorhynchus mykiss) RTG-2 cells. TBT treatment resulted in significant caspase activation, characteristic morphological changes, DNA fragmentation, and consequent apoptotic cell death in RTG-2 cells. TBT exposure induced the rapid and sustained accumulation of phosphorylated MAPKs, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38 MAPK). Further analysis using pharmacological inhibitors against caspases and MAPKs showed that TBT also induced cell death in a caspase-independent manner and that p38 MAPK is involved in TBT-induced caspase-independent cell death, whereas JNK is involved in the caspase-dependent apoptotic pathway. Thus, TBT employs at least two independent signaling cascades to mediate cell death in RTG-2 cells. To our knowledge, this is the first study revealing the relationship between MAPK activation and TBT cytotoxicity in RTG-2 cells.     

Conformation of the fowl protamine, galline, and its binding properties to DNA

1. CD spectra showed that the fowl protamine, galline, has an unordered structure rich in reverse turns in neutral solution. Eight reverse turns were predicted to be present in the galline molecule on the basis of its amino acid sequence. Spectrophotometric analyses revealed that galline efficiently bound to DNA in 0.25 mM EDTA/10 mM Tricine-HCl, pH 7.4, but hardly so in 30 mM NaCl/3 mM sodium citrate, pH 7.0. Citrate ions bound specifically to the galline molecule, causing a conformational change in it. As a result, galline could not interact with DNA. 2. The concentration of unbound galline in a mixture of DNA and galline in 100 mM NaCl/50 mM Tricine-HCl, pH 7.4, at 37 C was determined by measurement of the intrinsic fluorescence of tyrosine residues of galline in the supernatant after ultracentrifugation of the mixture. The Scatchard plot showed positive co-operativity in the binding of galline to DNA and the binding parameters were determined: the co-operative binding constant (Kc) = 3.3 X 10(7)M-1, the co-operativity factor (q) = 800, and the number of nucleotides of DNA occupied by one galline molecule (n) = 28. The Kc and q values were intermediate between those for clupeine Z from herring sperm and S-methyl protamine from boar sperm. That is, the binding constants of protamine as to DNA decrease in the order of herring, fowl, and boar, while the co-operativities in binding increase in that order.     

Diurnal change of tartrate dissimilation during the ripening of grapes

l(+)-tartrate-[U-¹⁴C] or sucrose-[U-¹⁴C] was fed into grape berries and ¹⁴CO₂ evolution was determined. ¹⁴CO₂ evolution front l(+)-tartrate-[U-¹⁴C] was slightly higher in mature than immature berries, and that from sucrose-[U-¹⁴C] was higher in immature than mature ones. ¹⁴CO₂ evolution from l(+)-tartrate-[U-¹⁴C] was irregular throughout the day until 2 or 3 weeks after flowering. This stage shifted to regular ¹⁴CO₂ evolution until 6 or 7 weeks after flowering, and the mode of ¹⁴CO₂ evolution showed diurnal variation; higher in the day than at night. Then the stage without variation of ¹⁴CO₂ evolution followed 10 weeks after flowering. These observations indicate that tartrate is not biochemically inert in grape berries, while the amount of ¹⁴CO₂ evolution from sucrose-[U-¹⁴C] was higher at night than in the day through the whole ripening process, except in the early stage.     

Reprogramming to a pluripotent state modifies mesenchymal stem cell resistance to oxidative stress

Properties of induced pluripotent stem cells (iPSC) have been extensively studied since their first derivation in 2006. However, the modification in reactive oxygen species (ROS) production and detoxification caused by reprogramming still needs to be further elucidated. The objective of this study was to compare the response of iPSC generated from menstrual blood–derived mesenchymal stem cells (mb‐iPSC), embryonic stem cells (H9) and adult menstrual blood–derived mesenchymal stem cells (mbMSC) to ROS exposure and investigate the effects of reprogramming on cellular oxidative stress (OS). mbMSC were extremely resistant to ROS exposure, however, mb‐iPSC were 10‐fold less resistant to H₂O₂, which was very similar to embryonic stem cell sensitivity. Extracellular production of ROS was also similar in mb‐iPSC and H9 and almost threefold lower than in mbMSC. Furthermore, intracellular amounts of ROS were higher in mb‐iPSC and H9 when compared with mbMSC. As the ability to metabolize ROS is related to antioxidant enzymes, we analysed enzyme activities in these cell types. Catalase and superoxide dismutase activities were reduced in mb‐iPSC and H9 when compared with mbMSC. Finally, cell adhesion under OS conditions was impaired in mb‐iPSC when compared with mbMSC, albeit similar to H9. Thus, reprogramming leads to profound modifications in extracellular ROS production accompanied by loss of the ability to handle OS.     

Emergence of complex rearrangements at translocation breakpoints in a transgenic mouse; implications for mechanisms involved in the formation of chromosome rearrangements

Cryptic complex rearrangements as a result of a reciprocal chromosome translocation have been characterised in a transgenic mouse strain. Analysis of the breakpoint junctions in our previous studies showed that the ada transgene was integrated at the breakpoint forming a fusion gene with Golga3 (Mea2). In this study, further detailed analysis around the translocation junctions revealed that the surrounding regions were composed of 13 fragments of defined transgenic chromosome origins over approximately 1.9-Mb areas. Exactly the same cluster structure of these 13 breakpoint fragments already existed in the second generation of the transgenic mice. Our results show that this highly complex rearrangement has been conserved as the incipient form without any additional changes for 18 years up to the present generation, suggesting simultaneous occurrence of multiple events in the founder mouse.     

Mechanism of dopachrome tautomerization into 5,6-dihydroxyindole-2-carboxylic acid catalyzed by Cu(II) based on quantum chemical calculations

Background

Tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) is a biologically crucial reaction relevant to melanin synthesis, cellular antioxidation, and cross-talk among epidermal cells. Since dopachrome spontaneously converts into 5,6-dihydroxyindole (DHI) via decarboxylation without any enzymes at physiologically usual pH, the mechanism of how tautomerization to DHICA occurs in physiological system is a subject of intense debate. A previous work has found that Cu(II) is an important factor to catalyze the tautomerization of dopachrome to DHICA. However, the effect of Cu(II) on the tautomerization has not been clarified at the atomic level.

Methods

We propose the reaction mechanism of the tautomerization to DHICA by Cu(II) from density functional theory-based calculation.

Results

We clarified that the activation barriers of α-deprotonation, β-deprotonation, and decarboxylation from dopachrome are significantly reduced by coordination of Cu(II) to quinonoid oxygens (5,6-oxygens) of dopachrome, with the lowest activation barrier of β-deprotonation among them. In contrast to our previous work, in which β-deprotonation and quinonoid protonation (O5/O6-protonation) were shown to be important to form DHI, our results show that the Cu(II) coordination to quinonoid oxygens inhibits the quinonoid protonation, leading to the preference of proton rearrangement from β-carbon to carboxylate group but not to the quinonoid oxygens.

Conclusion

Integrating these results, we conclude that dopachrome tautomerization first proceeds via proton rearrangement from β-carbon to carboxylate group and subsequently undergoes α-deprotonation to form DHICA.

General significance

This study would provide the biochemical basis of DHICA metabolism and the generalized view of dopachrome conversion which is important to understand melanogenesis.