Structure determination of inonotsuoxides A and B and in vivo anti-tumor promoting activity of inotodiol from the sclerotia of Inonotus obliquus

Two new lanostane-type triterpenoids, inonotsuoxides A (1) and B (2) along with three known lanostane-type triterpenoids, inotodiol (3), trametenolic acid (4), and lanosterol (5), were isolated from the sclerotia of Inonotus obliquus (Pers.: Fr.) (Japanese name: Kabanoanakake) (Russian name: Chaga). Their structures were determined to be 22R,25-epoxylanost-8-ene-3beta,24S-diol (1) and 22S,25-epoxylanost-8-ene-3beta,24S-diol (2) on the basis of spectral data including single crystal X-ray analysis. These compounds except for 2 were tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), as a test for potential cancer chemopreventive agents. The most abundant triterpene, inotodiol (3), was investigated for the inhibitory effect in a two-stage carcinogenesis test on mouse skin using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter. Compound 3 was found to exhibit the potent anti-tumor promoting activity in the in vivo carcinogenesis test.     

α-Amylase production is induced by sulfuric acid in rice aleurone cells

The hydrolytic enzyme alpha-amylase (EC 3.2.1.1) is produced mainly in aleurone cells of germinating cereals, and the phytohormone gibberellin (GA) is essential for its induction. However, in rice (Oryza sativa L.), sulfuric acid (H(2)SO(4)) induces alpha-amylase production in aleurone tissue even in the absence of GA. Here, the pre-treatment of rice aleurone cells with H(2)SO(4) and incubation in water induced alpha-amylase activity, as if the cells had been incubated in GA solution.     

SWAP-70 is required for oncogenic transformation by v-Src in mouse embryo fibroblasts

SWAP-70 is a phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) binding protein, which acts in F-actin rearrangement. The role of SWAP-70 in oncogenic transformation of mouse embryo fibroblasts (MEFs) by v-Src was examined by use of MEFs defective in SWAP-70. v-Src morphologically transformed MEFs lacking SWAP-70, but growth of the transformed cells in culture was slower than that of cells supplemented with exogenous SWAP-70. The v-Src-transformed MEFs deficient in SWAP-70 were unable to grow in soft agar while those expressing SWAP70 readily formed colonies, suggesting that SWAP-70 is required for anchorage independent growth of v-Src transformed MEFs. When transplanted in nude mice, tumors formed by the v-Src transformed SWAP-70(-/-) MEFs were smaller than those formed by cells expressing exogenous SWAP-70. These results suggest that SWAP-70 may be required for oncogenic transformation and contributes to cell growth in MEFs transformed by v-Src.     

Cholesterol and lipid/protein ratio control the oligomerization of a sphingomyelin-specific toxin, lysenin

Lysenin is a pore-forming toxin that specifically binds sphingomyelin (SM). The binding of the toxin to the membrane is accompanied by the oligomerization of the protein, leading to pore formation. The interaction of lysenin with SM is affected by the presence of other lipids found in the plasma membrane. Although a previous study showed that SM/cholesterol liposomes were 10,000 times more effective than SM liposomes in inhibiting lysenin-induced hemolysis (Yamaji, A., Sekizawa, Y., Emoto, K., Sakuraba, H., Inoue, K., Kobayashi, H., and Umeda, M. (1998) J. Biol. Chem. 273, 5300-5306), the role of cholesterol is not precisely clarified. In the present study, we examined the effects of the presence of cholesterol in the SM membrane on the inhibition of hemolysis, the binding of lysenin to SM, and the oligomerization of lysenin. The addition of cholesterol to SM liposomes dramatically inhibited lysenin-induced hemolysis as described previously. However, the presence of cholesterol did not affect the binding of lysenin to SM liposomes. The oligomerization of lysenin was facilitated by the presence of cholesterol in SM liposomes. The oligomerization of lysenin was also dependent on the SM/lysenin ratio, that is, the amount of lysenin oligomer was increased with the decrease in the SM/lysenin ratio. When the SM/lysenin molar ratio was high, lysenin associated with the membrane as a monomer, which was able to transfer to the erythrocyte membrane. Our results indicate that both cholesterol and the SM/lysenin ratio control the amount of lysenin monomer via altering the state of protein oligomerization, thus affecting hemolysis.     

Focal adhesion kinase determines the fate of death or survival of cells in response to TNFalpha in the presence of actinomycin D

We speculated that focal adhesion kinase (FAK) might play a critical role in the TNFalpha-induced cell death. In this study, we found that FAK-/- cells are more sensitive to TNFalpha-induced apoptosis in the presence of actinomycin D (Act D) compared to FAK+/- cells. Prosurvival pathways are activated by the rapid recruitment of complex I, comprising TNFR1, TRADD, RIP and TRAF2, which leads to the activation of the NF-kappaB pathway. On the other hand, proapoptotic pathways are activated by complex II, the death-inducing signaling complex (DISC), which contains TNFR1, TRADD, RIP, and FADD, and procaspase-8 proteins. As TNFR1, TRADD, and RIP are included in both Complex I and DISC, we speculated that RIP might be a key protein. Coimmunoprecipitation assays revealed that RIP is included in complex I in FAK+/- cells, and FAK was associated with RIP. On the other hand, RIP is included in DISC in FAK-/- cells. FAK might be a key protein in the formation of complex I and the activation of NF-kappaB. Furthermore, Akt was activated in FAK+/- cells, but not FAK-/- cells. In conclusion, we first demonstrated that FAK determines the pathway leading to death or survival in TNFalpha/ActD-stimulated fibroblasts.     

Protective effects of carotenoids from saffron on neuronal injury in vitro and in vivo

Crocus sativus L. (saffron) has been used as a spice for flavoring and coloring food preparations, and in Chinese traditional medicine as an anodyne or tranquilizer. Our previous study demonstrated that crocin, a carotenoid pigment of saffron, can suppress the serum deprivation-induced death of PC12 cells by increasing glutathione (GSH) synthesis and thus inhibiting neutral sphingomyelinase (nSMase) activity and ceramide formation. The carotenoid pigments of saffron consist of crocetin di-(beta-d-glucosyl)-ester [dicrocin], crocetin-(beta-d-gentiobiosyl)-(beta-d-glucosyl)-ester [tricrocin] and crocetin-di-(beta-d-gentiobiosyl)-ester [crocin]. Saffron also contains picrocrocin, the substance causing saffron's bitter taste. In this study, to confirm whether neuroprotective effects of saffron are caused solely by crocin, we examined the antioxidant and GSH-synthetic activities of these crocins in PC12 cells under serum-free and hypoxic conditions. Measurements of cell viability, peroxidized membrane lipids and caspase-3 activity showed that the rank order of the neuroprotective potency at a concentration of 10 muM was crocin>tricrocin>dicrocin and picrocrocin (the latter two crocins had a little or no potency). In addition, we show that among these saffron's constituents, crocin most effectively promotes mRNA expression of gamma-glutamylcysteinyl synthase (gamma-GCS), which contributes to GSH synthesis as the rate-limiting enzyme, and that the carotenoid can significantly reduce infarcted areas caused by occlusion of the middle cerebral artery (MCA) in mice.     

Characterization of Japanese flounder (Paralichthys olivaceus) NK-lysin, an antimicrobial peptide

The NK-lysin cDNA of Japanese flounder, Paralichthys olivaceus, consists of 657bp, containing an open reading frame (ORF) of 444bp, which encodes 147 amino acid residues. The amino acid sequence of Japanese flounder NK-lysin has 21% identity to porcine NK-lysin and bovine NK-lysin, 23% to equine NK-lysin, and 46% to zebrafish NK-lysin-like protein. Multiple alignments of Japanese flounder NK-lysin and other known saposin-like proteins revealed that the six cysteine residues important for structural folding are completely conserved. The Japanese flounder NK-lysin gene is approximately 2kb and consists of five exons and four introns. Japanese flounder NK-lysin mRNA constitutive expression was mainly detected in gills, heart, head kidney, intestines, peripheral blood leukocytes (PBLs), spleen and trunk kidney, and was detected at low levels in liver, muscle and ovary. However, expression was not detected in brain, skin and stomach of apparently healthy Japanese flounder. Gene expression of Japanese flounder NK-lysin was not inducible by lipopolysaccharide (LPS) treatment. A synthesized NK-lysin peptide, consisting of 27 amino acid residues, showed antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Photobacterium damselae subsp. piscicida.     

Paraliparis variabilidens,a new snailfish (Liparidae) from the Suruga Trough,Japan

Ichthyological Research - The new snailfish Paraliparis variabilidens is described from a single specimen collected between 1,462 and 1,562 m depth in the northern part of the Suruga...