Cell kinetics-dependent antitumor effect of irinotecan hydrochloride induced by the synchronizing effect of hydroxyurea: cell kinetics and dosing time

Influence of hydroxyurea (HU) on the antitumor effect of irinotecan hydrochloride (CPT-11) was investigated in ICR male mice transplanted with sarcoma 180 cells (S-180). A single dose of CPT-11 (100 mg/kg) was injected at various times after a single dose of HU (300 mg/kg). The relative tumor weight varied significantly depending on the timing of CPT-11 injection after HU injection (P < 0.01). The higher antitumor effect of CPT-11 was observed when DNA synthesis of S-180 cells increased (20 hr), and the lower effect was observed when the DNA synthesis decreased (0 hr). The loss of body weight also varied significantly depending on the timing of CPT-11 injection after HU injection (P < 0.01). The toxicity of CPT-11 was higher when the inhibitory effect of HU on DNA synthesis of bone marrow cells was stronger (15 hr), and the lower toxicity was observed when the inhibitory effect was not observed (0 hr). The plasma SN-38 concentration at 2 hr after CPT-11 injection was higher at 20 hr after HU injection than at 0 hr after HU injection. The difference in plasma esterase activity between 0 hr and 20 hr after HU injection was regarded as the mechanism underlying the dosing time-dependent difference of the SN-38 concentration. These experiments suggest that HU can produce a different phase of cell cycle between tumor cells and normal cells. This leads to increase the antitumor effect of CPT-11 without increasing the adverse effect of the drug. It is essential to consider the dosing time in the two-drug combination therapy.     

Comparing areas of suitable habitats along travelled and possible shortest routes in migration of White-naped Cranes Grus vipio in East Asia

Many migratory species take detours when migrating from their breeding to wintering grounds rather than following the shortest route available. To test whether the distribution of potentially suitable habitats might be a factor causing the use of less direct detours during migration, we analysed the migratory routes of five White-naped Cranes Grus vipio satellite-tracked from central–east Russia, and compared the total area of wetlands and grasslands along the migratory routes travelled by the Cranes with that along the shortest possible routes to the Cranes' wintering grounds. All five Cranes made an easterly detour, and the distance ratio of the routes used by Cranes to the shortest possible route was 1.13 ± 0.03 sd. Based on National Oceanic and Atmospheric Administration satellite images, we demonstrate that the area of wetlands and grasslands along the migration routes travelled by Cranes was greater than along the shortest possible routes.     

Siglec-9 modulated IL-4 responses in the macrophage cell line RAW264

Siglecs, an immunoglobulin-like lectin family that recognizes the sialic acid moiety, regulate various aspects of immune responses. In the present study, we investigated the effects of Siglecs on the macrophage cell line RAW264, which was stimulated with interleukin-4 (IL-4). The induction of arginase-1 (Arg1) by IL-4 was stronger in Siglec-9-expressing cells than in mock cells. Mutations in the cytoplasmic tyrosine-based inhibitory motifs in Siglec-9 markedly reduced the expression of Arg1. The phosphorylation of Akt by IL-4 and extracellular signal-regulated kinase (ERK) without IL-4 was stronger in Siglec-9-expressing cells, indicating the enhanced activation of the phosphatidylinositol 3 kinase (PI-3K) and mitogen-activated protein kinase kinase (MEK)/ERK pathways, respectively. The enhanced expression of Arg1 was inhibited by MEK inhibitors, but not by PI-3K inhibitor. These results indicate that Siglec-9 affects several different signaling pathways in IL-4-stimulated macrophages, which resulted in enhanced induction of Arg1 in Siglec-9-expressing RAW264 cells.     

Inhibition of the integrin signal constitutes a mouse iPS cell niche

Stem cells are regulated by their surrounding microenvironments, called niche, such as cell–cell interaction and extracellular matrix. Classically, feeder cells as a niche have been used in the culture of iPS cells from both the mouse and the human. However, the regulation mechanism of stem cells by feeder cells as a niche still have been partially unclear. In this study, we used three murine iPS cell lines, iPS‐MEF‐Ng‐20D‐17, iPS‐MEF‐Ng‐178B‐5 and iPS‐MEF‐Fb/Ng‐440A‐3, which were generated by different reprogramming methods. In general, these cell lines commonly need the feeder cells as a niche to culture. Recently, the effect of substrate stiffness is known in stem cell study. First, we focused on the mechanical properties of feeder cells, and then we speculated that feeder‐less culture might be made possible by using molecules in place of the mechanical properties of the niche. Finally, we found that the combination of disintegrin (echistatin) and 2i (GSK3 inhibitor and MEK inhibitor) is a sufficient condition for three murine iPS culture. This novel method of mimicking the murine iPS cell niche may be useful to understand signaling pathways to maintain the pluripotency of stem cells.     

Wind conditions facilitate the seasonal water‐crossing behaviour of Oriental Honey‐buzzards Pernis ptilorhynchus over the East China Sea

Migratory raptors rarely fly over stretches of water larger than 25 km, although different species undertake water crossings of varying lengths, depending mainly on their wing morphology. Oriental Honey‐buzzards fly c. 680 km over the East China Sea in autumn from breeding areas in Japan to wintering areas in Southeast Asia, but avoid this long water crossing in spring. We investigated the effects of weather on this exceptional migratory behaviour and its seasonality through a maximum entropy niche modelling approach. We used data collected through satellite tracking of 31 adult birds as presence points and a set of variables related to wind, precipitation and convective condition as environmental predictors. Results of modelling showed very different, almost non‐overlapping, areas suitable for migration over the East China Sea region in autumn and spring. Suitable migration routes in autumn mostly occurred over the sea, whereas suitable areas for spring migration mostly occurred over land, suggesting that circumnavigating the East China Sea is preferable in spring. At the regional scale, wind conditions facilitate water‐crossing behaviour of Oriental Honey‐buzzards in autumn, but not in spring. Specifically, suitable tailwinds over the sea enable water‐crossing in autumn, whereas in spring, wind support and convective conditions are best over land. Our modelling did not suggest any importance of convective conditions for autumn migration. However, we expect that at smaller temporal scales, convective conditions would be a considerable facilitator of the water‐crossing behaviour in this species.     

Up-regulation of ras-GAP genes is reversed by a MEK inhibitor and doxorubicin in v-Ki-ras-transformed NIH/3T3 fibroblasts

Ras-GTPase-activating proteins (Ras-GAPs) have been implicated both as suppressors of Ras and as effectors in regulating cellular activities. To study whether Ras-GAPs have roles in tumor cell survival or not, mRNA levels of ras-related genes were measured in v-Ki-ras-transformed (DT) and the parental NIH/3T3 cells, using real-time PCR. mRNA levels of p120-Gap, Gap1(m), and PIK3CA were increased in DT cells compared with NIH/3T3 cells. p120-Gap and PIK3CA genes were induced by addition of serum or epidermal growth factor to serum-starved DT cells. Three anti-cancer drugs, an ERK kinase (MEK) inhibitor PD98059, a topoisomerase II poison doxorubicin (adriamycin), and a histone deacetylase inhibitor trichostatin A, selectively blocked the overexpression of p120-Gap and Gap1(m) genes in DT cells. These drugs also caused reversion of DT cells to the adherent shape associated with growth arrest. Our results suggest that p120-Gap and Gap1(m) genes provide important biomarkers for cancer therapies.     

Interspecies compatibility of NAS1 gene promoters.

Nicotianamine and nicotianamine synthase (NAS) play key roles in iron nutrition in all higher plants. However, the mechanism underlying the regulation of NAS expression differs among plant species. Sequences homologous to iron deficiency-responsive elements (IDEs), i.e., cis-acting elements, are found on the promoters of these genes. We aimed to verify the interspecies compatibility of the Fe-deficiency response of NAS1 genes and understand the universal mechanisms that regulate their expression patterns in higher plants. Therefore, we introduced the graminaceous (Hordeum vulgare L. and Oryza sativa L.) NAS1 promoter::GUS into dicots (Nicotiana tabacum L. and Arabidopsis thaliana L.). Fe deficiency induced HvNAS1 expression in the shoots and roots when introduced into rice. HvNAS1 promoter::GUS and OsNAS1 promoter::GUS induced strong expression of GUS under Fe-deficient conditions in transformed tobacco. In contrast, these promoters only definitely functioned in Arabidopsis transformants. These results suggest that some Fe nutrition-related trans-factors are not compatible between graminaceous plants and Arabidopsis. HvNAS1 promoter::GUS induced GUS activity only in the roots of transformed tobacco under Fe-deficient conditions. On the other hand, OsNAS1 promoter::GUS induced GUS activity in both the roots and shoots of transformed tobacco under conditions of Fe deficiency. In tobacco transformants, the induction of GUS activity was induced earlier in the shoots than roots. These results suggest that the HvNAS1 and OsNAS1 promoters are compatible with Fe-acquisition-related trans-factors in the roots of tobacco and that the OsNAS1 promoter is also compatible with some shoot-specific Fe deficiency-related trans-factors in tobacco.     

A versatile strategy for the synthesis of crown ether-bearing heterocycles: discovery of calcium-selective fluoroionophore

Heterocycles have been modified in various ways in the search for new functions, but few examples are known of crown ethers incorporating heterocycles in macro-ring systems. Here we report a simple and versatile synthesis of crown ether-bearing heterocycles. An acylurea moiety in the heterocycles is efficiently transformed to 'crown ether' of various ring sizes. The products included a Ca(2+)-selective fluoroionophore. Our simple methodology is expected to provide many novel functional heterocyclic compounds, including fluoroionophores and candidate pharmaceuticals.     

Upregulation of IGF-I in the goldfish retinal ganglion cells during the early stage of optic nerve regeneration

Goldfish retinal ganglion cells (RGCs) can regrow their axons after optic nerve injury. However, the reason why goldfish RGCs can regenerate after nerve injury is largely unknown at the molecular level. To investigate regenerative properties of goldfish RGCs, we divided the RGC regeneration process into two components: (1) RGC survival, and (2) axonal elongation processes. To characterize the RGC survival signaling pathway after optic nerve injury, we investigated cell survival/death signals such as Bcl-2 family members in the goldfish retina. Amounts of phospho-Akt (p-Akt) and phospho-Bad (p-Bad) in the goldfish retina rapidly increased four- to five-fold at the protein level by 3-5 days after nerve injury. Subsequently, Bcl-2 levels increased 1.7-fold, accompanied by a slight reduction in caspase-3 activity 10-20 days after injury. Furthermore, level of insulin-like growth factor-I (IGF-I), which activates the phosphatidyl inositol-3-kinase (PI3K)/Akt system, increased 2-3 days earlier than that of p-Akt in the goldfish retina. The cellular localization of these molecular changes was limited to RGCs. IGF-I treatment significantly induced phosphorylation of Akt, and strikingly induced neurite outgrowth in the goldfish retina in vitro. On the contrary, addition of the PI3K inhibitor wortmannin, and IGF-I antibody inhibited Akt phosphorylation and neurite outgrowth in an explant culture. Thus, we demonstrated, for the first time, the signal cascade for early upregulation of IGF-I, leading to RGC survival and axonal regeneration in adult goldfish retinas through PI3K/Akt system after optic nerve injury. The present data strongly indicate that IGF-I is one of the most important molecules for controlling regeneration of RGCs after optic nerve injury.