Inhibition of epidermal growth factor-induced DNA synthesis by tyrosine kinase inhibitors

We prepared methyl 2,5-dihydroxycinnamate as a stable analogue of erbstatin, a tyrosine kinase inhibitor. This analogue was about 4 times more stable than erbstatin in calf serum. It inhibited epidermal growth factor receptor-associated tyrosine kinase in vitro with an IC₅₀ of 0.15 μg/ml. It also inhibited in situ autophosphorylation of epidermal growth factor receptor in A431 cells. Methyl 2,5-dihydroxycinnamate was shown to delay the S-phase induction by epidermal growth factor in quiescent normal rat kidney cells, without affecting the total amount ofDNA synthesis. The effect of erbstatin on S-phase induction was smaller, possibly because of its shorter life time.     

Induction of multiple embryos with NaHCO3 or calcium free sea water in the horseshoe crab

Summary When horseshoe crab embryos were treated with NaHCO₃ at the developmental stage when the germ disc appears, multiple embryos were formed. NaHCO₃ may effect the formation of multiple embryos by binding Ca²⁺ ions of the embryo since multiple embryos were also formed by treatment with Ca²⁺ free sea water.The treatment caused the blastoderm layer to tear. When the embryos were returned to normal sea water after the treatment, the blastoderm recovered. Some cell masses, probably derived from the germ disc or its prospective cells, formed during the process of the recovery. Each cell mass developed into an embryo.Contributions from the Shimoda Marine Research Center, Nor. 348     

Performance of affinity chromatography columns

Performance of affinity chromatography columns was studied by measuring the rates of adsorption and elution of trypsin in a Sepharose 4B-soybean trypsin inhibitor column and a Sepharose 4B-arginine peptides column. The volumetric coefficient for trypsin transfer was evaluated from the break-through curves of trypsin, and elution profiles bed height of Sepharose 4B-STI column was estimated based on these results.     

Investigation of telomere length dynamics in induced pluripotent stem cells using quantitative fluorescence in situ hybridization

Here we attempted to clarify telomere metabolism in parental cells and their derived clonal human induced pluripotent stem cells (iPSCs) at different passages using quantitative fluorescence in situ hybridization (Q-FISH). Our methodology involved estimation of the individual telomere lengths of chromosomal arms in individual cells within each clone in relation to telomere fluorescence units (TFUs) determined by Q-FISH. TFUs were very variable within the same metaphase spread and within the same cell. TFUs of the established iPSCs derived from human amnion (hAM933 iPSCs), expressed as mean values of the median TFUs of 20 karyotypes, were significantly longer than those of the parental cells, although the telomere extension rates varied quite significantly among the clones. Twenty metaphase spreads from hAM933 iPSCs demonstrated no chromosomal instability. The iPSCs established from fetal lung fibroblasts (MRC-5) did not exhibit telomere shortening and chromosomal instability as the number of passages increased. However, the telomeres of other iPSCs derived from MRC-5 became shorter as the number of passages increased, and one (5%) of 20 metaphase spreads showed chromosomal abnormalities including X trisomy at an early stage and all 20 showed abnormalities including X and 12 trisomies at the late stage.     

Xylitol Production by an Enterobacter Species

A xylose-utilizing bacterial strain was isolated from soil.

The strain, No. 553, was identified as Enterobacter liquefaciens from the result of the taxonomical studies. This bacterium grew well on D-xylose as a sole carbon source and accumulated pentitol extracellularly in shaking culture.

Pentitol produced was isolated from the culture broth and identified as xylitol.

The xylitol production reached the maximum after the cessation of the cell growth with a yield of 33.3 mg per ml in a medium containing 10% D-xylose as a sole carbon source and no significant decline of the amount of xylitol was observed through the period of the cultivation.     

Studies on BHC Isomers and their Related Compounds

β-BTC(3, 4/5, 6),¹⁾ γ-BTC(3, 4, 6/5), and ε-BTC(3, 4, 5/6) were synthesized from α-BTC (3, 6/4, 5) by stepwise routes.     

Isolation and Characterization of PDE-I and II,the Inhibitors of Cyclic Adenosine-3′,5′-monophosphate Phosphodiesterase

In the screening for inhibitors of cyclic adenosine-3′,5′-monophosphate phosphodiesterase, two compounds, PDE-I (C₁₃H₁₃N₃O₅) and PDE-II (C₁₄H₁₄N₂O₅), were isolated from culture filtrates of a Streptomyces. Concentrations for 50% inhibitions of PDE-I and PDE-II against the high Km enzyme were 15 µm and 13 µm, and those against the low Km enzyme were 65 µm and 130 µm, respectively. Production, isolation and characterization of these compounds are described.     

Studies on Glyoxalase Inhibitor Isolation of a New Active Agent,MS-3, from a Mushroom Culture

Cultured broths were screened by measuring the inhibition of glyoxalase, using 105,000 g supernatant of rat liver homogenates as a crude enzyme. An active agent, MS–3 (C₂₁H₂₄O₇), was isolated from a cultured mushroom. ID₅₀ value of MS–3 against the crude glyoxalase was 12 mcg/ml. MS–3 has low toxicity and inhibited the growth of Yoshida rat sarcoma cells in cell culture. The production, isolation, and properties of MS–3 are described.     

The Reaction Mechanism of Rat Liver Glyoxalase I and Its Inhibition by MS-3

Glyoxalase I from rat liver was purified about 25-fold by acetone fractionation and ion-exchange chromatography on CM-Sephadex and DEAE-cellulose columns. The kinetic study of the enzymatic reaction supported the one-substrate mechanism : the hemimercaptal adduct produced nonenzymatically from methylglyoxal and glutathione is the substrate. The Km value determined was 0.1 mm and similar to that of porcine erythrocytes enzyme but differed significantly from that of yeast enzyme. It was inhibited by free glutathione competitively (Ki 1.2 mm). Kinetic studies on inhibition of glyoxalase I by MS–3 which was obtained from a cultured mushroom, Stereum hirsutum, indicated the inhibition type was competitive with the hemimercaptal adduct (Ki 4.6 × 10^?6 m). By the graphical study of the multiple inhibition kinetics free glutathione and MS–3 were shown to bind at the same sites of the enzyme.