TAE226, a Bis-Anilino Pyrimidine Compound,Inhibits the EGFR-Mutant Kinase Including T790M Mutant to Show Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells

TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation.     

Assessing temporal dynamics of predation and effectiveness of caterpillar visual defense using sawfly larval color and resting posture as a model

Caterpillars (Lepidoptera and Symphyta larvae) employ diverse visual defensive tactics, and effectiveness of such tactics may be highly dynamic across time due to seasonal changes in the predator assemblages and their preferences. However, this has rarely been studied especially in tropical regions. Here we assessed temporal changes in the defensive value of caterpillar color and shape, using six types of plasticine dummy caterpillars: three colors (green, black, and white) × two shapes (curled and straight). These dummy caterpillars were deployed five times over different seasons in tropical forests of Xishuangbanna (China) and, as a comparison, twice in a temperate forest of Hirosaki (Japan). The colors and shapes of dummy caterpillars simulate visual traits of black sawfly larvae which take the curled resting posture in tropical rainforests of Xishuangbanna, apparently masquerading excrements commonly found on plants, while in Hirosaki there is no black-curled sawfly larvae and few excrements on plants. We found no significant effects of caterpillar colors or shapes on predation in Hirosaki. In contrast, black and curled caterpillars received significantly lower predation by birds in Xishuangbanna constantly across time. However, we were unable to provide evidence that the black-curled sawfly larvae are masquerading as excrements. Shapes of the dummy caterpillars also affected the predation by ants and parasitoid wasps at certain times. This is the first report on ecological function of the curled posture of sawfly larvae, and we demonstrated the importance to assess the temporal dynamics of predation and effectiveness of defensive tactics in tropical forests.     

Effect of photoperiod and temperature on the induction of diapause in a Japanese strain of <Emphasis Type="Italic">Aphidoletes aphidimyza</Emphasis> (Diptera: Cecidomyiidae)

The aphidophagous gall midge Aphidoletes aphidimyza (Rondani) (Diptera: Cecidomyiidae), a dominant natural enemy of aphids, has been used as a biological control agent in many countries to control aphids in greenhouses. As developmental arrest in diapause lowers the effectiveness of natural enemies, we studied the effect of photoperiod and temperature on the incidence of diapause in a Japanese strain of A. aphidimyza by examining diapause induction under different day-length conditions in the laboratory. The critical day length for diapause induction was determined to be 12.7 h at 20°C. Diapause incidence was completely prevented at 30°C even though the photoperiod used was 11L13D. We also examined diapause induction under changing temperature conditions while maintaining the critical day length (12.7L11.3D). Diapause incidence was 100% in both field and greenhouse conditions under alternating temperatures of 20/16 or 25/16°C while the critical day length of 12.7 h was maintained. The Japanese strain of A. aphidimyza was sensitive to diapause entry from the first to the third noncocooned instar larval stages. Its eggs do not seem to be sensitive to diapause induction. Our results suggest that constant short-day conditions for at least four days are needed to induce diapause in the Japanese strain of A. aphidimyza.     

Antagonistic effect of disulfide-rich peptide aptamers selected by cDNA display on interleukin-6-dependent cell proliferation

Several engineered protein scaffolds have been developed recently to circumvent particular disadvantages of antibodies such as their large size and complex composition, low stability, and high production costs. We previously identified peptide aptamers containing one or two disulfide-bonds as an alternative ligand to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in length) were screened from a random peptide library by in vitro peptide selection using the evolutionary molecular engineering method "cDNA display". In this report, the antagonistic activity of the peptide aptamers were examined by an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an IL-6-dependent cell proliferation assay. The results revealed that a disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with similar efficacy to an anti-IL-6R monoclonal antibody.     

Characterization of the cysteine-rich calcium-binding S100A3 protein from human hair cuticles

S100A3, a unique protein among all members of the calcium-binding S100 family, is specifically expressed at the inner endocuticle of human hair fibers. Upon hair damage, S100A3 is released from hair fibers and possibly destabilizes the hair tissue architecture. This study describes the purification and characterization of native S100A3 isolated from human hair fibers. We extracted native S100A3 from cuticles and purified the protein by anion-exchange chromatography. The results of 2D gel electrophoresis showed that cuticle S100A3 has a slightly lower isoelectric point compared to the recombinant protein. Tandem mass spectrometry of the peptides resulting from endoproteinase digest of cuticle S100A3 revealed that the N-terminal methionine is replaced with an acetyl group. This is the first report on biochemical characteristics of S100A3 in hair cuticle.     

Detailed analysis of formation of chicken pituitary primordium in early embryonic development

The primordium of the mammalian adenohypophysis derived from Rathke's pouch (RP) is known to be formed by oral ectoderm invagination. However, in the early phase of pituitary development, the detailed process by which the oral ectoderm develops into the adenohypophysis remains largely unknown. Using high-resolution non-radiolabeled in situ hybridization and the BrdU and TUNEL methods, we have examined the detailed expression pattern of factors involved in the formation of RP of chicken and the changes in the mitotic and apoptotic cell regions in RP. In the chicken embryo, Sonic hedgehog (Shh) mRNA was initially expressed in the stomodeal plate but not in the oral ectoderm. After prospective diencephalon had detached from the oral ectoderm, another Shh-expressing region appeared in the most rostral part of the recess. LIM homeobox gene 3 (Lhx3) mRNA first appeared in the anterior area of Rathke's recess, and expression then spread to the caudal region. alphaGSU mRNA-expressing cells were observed at both ends of the Lhx3-expressing region, and thereafter the expression area moved to the posterior region. Furthermore, a close overlap was found between the proliferating region and Lhx3 mRNA-expressing area, and TUNEL-positive cells appeared in Seessel's pouch derived from the foregut. Thus, the primordium of the pituitary gland corresponding to the Lhx3-expressing region is surrounded by the Shh-expressing region, which appears in two steps, and the mass growth and invagination of RP of chicken result from the coordination of the dorsal extension of the anterior region and the ventral extension of the posterior region of RP.     

Changes in Crassulacean Acid Metabolism of Kalanchoe blossfeldiana by Different Nitrogen Sources

Kalanchoe blossfeldiana Poelln. cv. Hikan (a Crassulacean acidmetabolism (CAM) plant) was grown in pots containing soil for6 months and then cultured in nutrient solution containing 10mM nitrate or ammonium as a sole nitrogen source for 2 or 3months, under a long-day (16 h) condition. Plant growth was better in the nitrate medium. Leaves of thenitrate-grown plants showed greater diurnal fluctuations intitratable acidity and malate content than those of the ammonium-grownplants. The diurnal patterns in CO₂ exchange of nitrate-grownplants were basically similar for both groups, but the amountof net CO₂ uptake at night was twice as large in the nitrate-grownplants. The leaves of the nitrate-grown plants had 1.3 to 2.5times higher activities of phosphoenolpyruvate carboxylase (PEPC),phosphofructokinase (PFK) and NAD glycelaldehyde-3-phosphatedehydrogenase (G3PDH). These results indicate that K. blossfeldianagrown in nitrate medium showed more CAM activity than thosein ammonium medium. (Received August 13, 1987; Accepted February 22, 1988)     

Independent differentiation of mammotropes and somatotropes in the chicken embryonic pituitary gland

It has been reported that mammotropes in a rodent pituitary gland are derived from somatotropes via somatomammotropes (SMTs), cells that produce both growth hormone (GH) and prolactin (Prl). However, no studies have been done on the transdifferentiation of somatotropes in the chicken pituitary gland. In this study, in order to determine the origin of mammotropes, we studied detail property of appearance of chicken somatotropes, mammotropes and pit-1 cells and then evaluated the existence of SMTs in the chicken embryonic pituitary gland. Immunohistochemical analysis revealed that GH-immunopositive (GH-ip) cells appeared on embryonic day (E) 14 and were mainly distributed in the caudal lobe, while Prl-immunopositive (Prl-ip) cells appeared in the cephalic lobe of the pituitary gland on E16. In situ hybridization (ISH) and RT-PCR analysis showed that expression of GH and Prl mRNA starts at E12 in the caudal lobe and at E14 in the cephalic lobe respectively. Pit-1 mRNA was first detected on E5 by RT-PCR, and pit-1 mRNA-expressing cells were found in the cephalic lobe on E8. Then with the ontogeny of the chicken, these cells spread into both lobes. Using a double staining method with ISH and immunohistochemistry, we could not detect the existence of SMTs in the chicken embryonic pituitary gland even in the marginal region of either lobe. These results suggest that chicken somatotropes and mammotropes independently appear in different lobes of pituitary gland and that transdifferentiation from somatotropes to mammotropes is not the central route for differentiation of mammotropes in the embryonic chicken pituitary gland.     

Short-term ascorbic acid deficiency induced oxidative stress in the retinas of young guinea pigs

We examined whether short-term ascorbic acid deficiency induces oxidative stress in the retinas of young guinea pigs. Four-week-old guinea pigs were given a scorbutic diet (20 g/animal/day) with and without adequate ascorbic acid (400 mg/animal/day) in drinking water for 3 weeks. The serum concentrations of the reduced form of ascorbic acid and the oxidized form of ascorbic acid in the deficient group were 14.1 and 4.1%, respectively, of those in the adequate group. The retinal contents of the reduced form of ascorbic acid and the oxidized form of ascorbic acid in the deficient group were 6.4 and 27.3%, respectively, of those in the adequate group. The retinal content of thiobarbituric acid-reactive substances, an index of lipid peroxidation, was 1.9-fold higher in the deficient group than in the adequate group. Retinal reduced glutathione and vitamin E contents in the deficient group were 70.1 and 69.4%, respectively, of those in the adequate group. This ascorbic acid deficiency did not affect serum thiobarbituric acid-reactive substances and reduced glutathione concentrations but increased serum vitamin E concentration. These results indicate that short-term ascorbic acid deficiency induces oxidative stress in the retinas of young guinea pigs without disrupting systemic antioxidant status.