Toxicity of Jegosaponins A and B from Styrax japonica Siebold et al. Zuccarini in Prostate Cancer Cells and Zebrafish Embryos Resulting from Increased Membrane Permeability

(1) Background: Screening of medicinal herbs is one of the most powerful approaches to identifying novel therapeutic molecules against many human diseases. To avoid potential harmful effects during medicinal use, toxicity testing is necessary in the early stages of drug discovery. The objective of this study was to identify the cytotoxic mechanisms of jegosaponin A and B from Styrax japonica Siebold et al. Zuccarini; (2) Methods: We screened Japanese medicinal herb extracts using PC-3 prostate cancer cells and found that a methanol extract isolated from the unripe fruit of Styrax japonica Siebold et al. Zuccarini (SJSZ) had an inhibitory effect on cell viability. We further performed fractionation assays with PC-3 cells and identified the bioactive compounds using LC/MS and NMR analysis. We clarified the toxic mechanisms of these compounds using PC-3 cells and zebrafish embryos; (3) Results: We identified two active molecules, jegosaponin A and jegosaponin B, in the inhibitory fractions of the methanol extract. These jegosaponins are toxic to zebrafish embryos during the early developmental stage. Jegosaponin A and B showed strong haemolytic activity in sheep defibrinated blood (EC₅₀ = 2.1 μM, and 20.2 μM, respectively) and increased the cell membrane permeability in PC-3 cells and zebrafish embryos, which were identified using a membrane non-permeable DRAQ7, a fluorescent nucleus staining dye; (4) We identified the cytotoxic compounds jegosaponin A and B from SJSZ, which we showed to exhibit cell membrane disruptive properties using cell- and zebrafish-based testing.     

PLASIU: a system that facilitates creative decision-making in job-hunting

Through an analysis of university students’ job-hunting logs, we have found that their introspection via rereading their log sometimes helps them discover themselves. Then we have built a system called PLASIU designed to support job-hunter’s creative decision-making based on the observations from their actual job-hunting process. This paper provides an overview of PLASIU and describes the findings from a user study using PLASIU.     

Tubular hydroxyapatite formation through a hydrothermal process from α-tricalcium phosphate with anatase

Hydroxyapatite (HA) is a useful substance because of its biocompatibility and adsorption capability. Tubular HA particles are highly attractive as novel tissue scaffolds and as drug carriers because of their hollow structures. We previously found that tubular HA particles were formed when a mixture of α-tricalcium phosphate (α-TCP) and anatase was hydrothermally treated. However, the formation mechanism is still unclear. In this study, we investigated the effect of anatase on the formation of tubular HA particles. The formation of tubular HA particles was enhanced when a pellet of a mixture of α-TCP and anatase was UV-irradiated before hydrothermal treatment. The tubular HA formation was observed only when anatase particles with adequate size were added in adequate quantities. We found that the surface properties, size, and quantity of the anatase particles were important for the formation of tubular HA particles. The tubular HA particles were not formed in the early stages of the reaction, they were formed only after some crystal growth had occurred. The anatase particles controlled the nucleation and crystal growth of HA.     

Proteomic profiling of differential display analysis for human oral squamous cell carcinoma: 14-3-3 σ Protein is upregulated in human oral squamous cell carcinoma and dependent on the differentiation level

Oral squamous cell carcinoma (OSCC) has an absolute majority of all oral cancer. We used proteomic technology to analyze the protein expression profile in OSCC tissues and accompanying surrounding normal tissues in four oral locations (buccal mucosa, gingival mucosa, oral floor, and tongue). Ten protein spots were overexpressed more strongly in cancer tissues than normal ones, and were identified as proliferating cell nuclear antigen, 14-3-3 ε, 14-3-3 σ, proteasome subunit α type 5, translationally controlled tumor protein, eukaryotic translation initiation factor 3 subunit, macrophage capping protein, and mitochondrial isocitrate dehydrogenase subunit α. Macrophage capping protein and mitochondrial isocitrate dehydrogenase subunit α had two spots. Especially, we focused on 14-3-3 σ protein, one of the eight identified proteins, and assessed its expression level in four oral locations of OSCC by using differential display methods. The expression level of 14-3-3 σ protein was upregulated in four locations of oral cavity. Eight proteins which we identified in this study may play an important role in OSCC carcinogenesis and progression and could be used as diagnostic biomarkers of OSCC.     

Surface properties of perfluoroalkylethyl acrylate/n-alkyl acrylate copolymers

Wetting behavior of perfluoroalkylethyl acrylate (FA)/n-alkyl acrylate (AA) copolymers with the various length of side chains of the AAs is discussed from a standpoint of surface molecular mobility. The copolymerization reactivity ratio indicates that these polymers are random copolymers. The surface properties were studied by measuring dynamic contact angle, static contact angle and freeze-dried X-ray photoelectron spectroscopy, and the bulk properties by wide-angle X-ray diffraction and differential scanning calorimetry. The advancing contact angles for water were independent of side-chain length of AAs and were almost constant at 120°. We have attributed this phenomenon to the orientation of perfluoroalkyl groups (R_f groups, C_xF_2x+1) in air, which is independent of side-chain length of AAs. On the contrary, the receding contact angles showed small values of about 45° when n numbers below 8 and increased when n numbers above 12. This can be explained as follows. High wettability during the receding process at the n numbers below 8 results from regression of R_f groups at the water–solid interface caused by minimization of the interface free energy. The low wettability during the receding process at the n numbers above 12 shows that R_f groups cannot regress due to its crystallization. This mechanism is also supported by other measurements. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 1741–1749, 1999     

Effects of phorbol esters,A23187 and vasopressin on oleate metabolism in isolated rat hepatocytes

Studies were conducted to compare the metabolic effects of vasopressin, 4β-phorbol-12-myristate-13-acetate (PMA) and A23187 on ketogenesis and oleate metabolism in isolated hepatocytes from fed rats. Vasopressin inhibited the formation of acid-soluble products from [1-¹⁴C]oleate (0.25 mM, 0.5 mM and 1 mM), the inhibition being most marked at low (0.25 mM) concentration of oleate. Conversion of [1-¹⁴C]oleate into¹⁴CO₂ and esterified products was stimulated by vasopressin. The stimulatory effect of this hormone on¹⁴CO₂ production was most marked at high (1 mM) concentration of oleate, whereas that on [1-¹⁴C]-oleate esterification was most marked at low (0.25 mM) concentration of oleate. These vasopressin actions were abolished when hepatocytes were incubated in the absence of calcium in the medium. Our results strongly suggest that both increase in esterification and increase in oxidation to CO₂ contribute to the anti-ketogenic action of vasopressin when oleate is added as substrate, although the relative extent of their contribution varies according to the oleate concentration. The anti-ketogenic action of vasopressin was mimicked by PMA but not by A23187. PMA also caused a stimulation of [1-¹⁴C]oleate esterification although the effect was diminished at 1 mM [1-¹⁴C]oleate. A23187 failed to affect [1-¹⁴C]oleate esterification. The metabolic effects of PMA were elicited in the absence of extracellular calcium, too. Conversion of [1-¹⁴C]oleate into¹⁴CO₂ was only slightly increased by both PMA and A23187 when 1 mM [1-¹⁴C]oleate was added as substrate. The marked stimulatory effect of vasopressin on¹⁴CO₂ production from [1-¹⁴C]oleate was not reproduced even by the combination of PMA and A23187. The possible involvement of protein kinase C and calcium mobilization in the regulation of oleate metabolism is discussed.     

Anti-angiogenic effect of an insertional fusion protein of human basic fibroblast growth factor and ribonuclease-1

Human pancreatic ribonuclease-1 (RNase1) does not exhibit its cytotoxicity unless it is artificially internalized into the cytosol. Furthermore, once it encounters the cytosolic RNase inhibitor (RI), the activity of RNase1 is seriously reduced. To achieve the cellular targeting of RNase1 and the blocking of RI binding simultaneously, the basic fibroblast growth factor (bFGF) sequence was inserted into RNase1 at the RI binding site using a gene fusion technique. The effect of this fusion protein, CL-RFN89, on the angiogenesis, which was accelerated by FGF-FGF receptor interaction, was investigated. It was shown by using fluorescein-labeled CL-RFN89, that the binding to human umbilical vein endothelial cells (HUVECs) was dependent on the existence of the FGF receptors. In addition, CL-RFN89 inhibited the cellular growth of HUVECs in vitro and also inhibited the tube formation, using a three-dimensional tube formation assay. Furthermore, this fusion protein was shown to prevent in vivo tumor cell-induced angiogenesis, using the mouse dorsal air sac assay. These results demonstrated that CL-RFN89 inhibits angiogenesis in vitro and in vivo and that it can be expected to be a potent antiangiogenic agent.