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Based on the zymography analysis, Bacillus sp. DJ-4 (screened from Doen-Jang, a Korean traditional fermented food) secretes seven extracellular fibrinolytic enzymes (EFEs; 68, 64, 55, 45, 33, 27, and 13 kDa) in culture broth. These seven EFEs were analyzed by newly applied SDSfibrin zymography combined with gradient polyacrylamide (SDS-FZGP). This improved gel system was used with a 5-20% acrylamide gradient in a fibrin zymogram gel for the separation of proteins with molecular masses from below 10 kDa to over 100 kDa on one gel plate. Using this system, high molecular weight bands (HMWBs) were clearly and sharply resolved. We also examined the relationship between an acrylamide concentration and the enzymatic activity of EFE using densitometric analysis.

A 2-stage in vi tro tuberization pro cess com pris ing first micropropagation via nodal explants and then tu ber in duc tion in the re sul tant in vi tro plantlets was stud ied us ing 2 cultivars of po tato, Iwa and Daeji. In par tic u lar, the ef fects on both plantlet growth and sub se quent in vi tro tuberization of Murashige and Skoog (1962) basal me dium con tain ing ei ther su crose or malt ose, each at 3 % (w/v), used for micropropagation were in ves ti gated. Su crose and malt ose were found to be equally ef fec tive in sup port ing de vel op ment of vig or ous plantlets from the nodal explants of both po tato cultivars. Upon trans fer to a me dium with an op ti mised level of su crose (i.e. 8 %, w/v) for in vi tro tuberization, only the plantlets pre vi ously grown in the su crose-containing me dium were ca pa ble of form ing more microtubers of the larger size cat e gory (greater than 0.5 g). The rel a tive im por tance of sucrose sup ply at the mircropropagation stage was fur ther confirmed when the re sul tant plantlets grown in the 3 % sucrose-containing me dium were trans ferred to an in vi tro tuberization me dium con tain ing ei ther su crose or malt ose, each at 8 % (w/v). In this ex per i ment, malt ose and su crose had indistingushable ef fects on in vi tro tuberization. In tro duc tionSu crose has been shown to be a very im por tant factor for po tato in vi tro tuberization. For in stance, in a two-step in vi tro tuberization pro cess com pris ing first grow ing in vi tro po tato plantlets from nodal explants and then microtuber for ma tion by the resul tant plantlets, 8 % (w/v) su crose alone in the tuberization me dium was found to be suf fi cient with out the need for any ex og e nous plant growth reg u la tors (Leclerc et al. 1994, Yu et al. 2000.The ef fect of sub sti tut ing su crose with malt ose on in vi tro tuberization has been stud ied (Khuri and Moorby 1995). It was found that a mix ture of 4 % (w/v) su crose and 4 % (w/v) malt ose was in fe rior to 8 % (w/v) su crose for in vi tro tuberization. However, the ef fect of malt ose alone in the tuberization me dium was not es tab lished. Fur ther more, in this study the nodal expants were placed right from the be gin ning of the 2-step tuberization pro cess on a me dium con tain ing the mix ture of dissacharides. Like most other stud ies on a two-step pro cess of microtuber for ma tion, there was no due re gard to the dis tinct na ture of the two steps in volved. As a con se quence, it is not pos si ble to de ter mine how 47 ACTA PHYSI OLO GIAE PLAN TA RUM

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Even though target-specific cancer therapies have some progress, only 5% of such therapy show efficacy in the clinic. Therefore, the development of cancer therapy targeted to cytotoxicity is still important. Cell cycle control is the major regulatory mechanism of cell growth. Small chemicals that inhibit cell cycle progression have attracted much attention for cancer therapy because they can block tumor growth. Drug-mediated mitotic-checkpoint-dependent-arrest is often followed by cell death. 2In previous studies, we reported a diaryl oxazole compound, KRIBB3 (1a) that displayed strong anti-mitotic activity against cancer cells.3 KRIBB3 showed inhibition of proliferation of HCT-116 colorectal cancer cells with GI 50 value of 0.1 μM and showed 6 times stronger inhibitory activity than nocodazol. Nocodazole is a well known anti-mitotic chemical and we used it as a control compound. In an effort to further develop this promising compound 1a toward a potent anti-cancer agent, we have focused on examining structure-activity relationships for KRIBB3. We synthesized a series of diaryl isoxazole derivatives by modifying alkoxy and hydroxyl group in the aryl moiety of KRIBB3 (1a), and evaluated their antiproliferative activity on the proliferation of HCT-116 colorectal cancer cells. Herein, we describe the synthesis and biological evaluation of KRIBB3 analogues 1.The synthesis of diaryl isoxazole derivative 1b was achieved through the route in Scheme 1. The formylation 4 of 4-ethylresorcinol (2) with DMF and POCl 3 gave 5-ethyl-2-hydroxy-4-methoxybenzaldehyde which was dimethylated utilizing methyl iodide and potassium carbonate to furnish compound 3. Reaction of aldehyde 3 with 4-methoxybenzylmagnesium chloride and the subsequent oxidation of the resulting secondary alcohol with TPAP and NMO afforded ketone 4.5 Condensation of compound 4 with dimethylformamide dimethylacetal (DMFDMA) in refluxing toluene gave enaminoketone 5, 6 which was cyclized in refluxing methanolic AcOH in the presence of Na 2 CO 3 to provide isoxazole 1b.6b Compound 1c and 1f were prepared from 2-benzyloxy-4-methoxy-5-ethylbenzaldehyde and 3,5-dimethyl-4-hydroxybenzaldehyde, respectively, in same fashion as described for synthesis of 1b. Compound 1d and 1e were prepared by demethylation of 1c using BBr3 in 45% and 30% yields, repectively. Compound 1g was synthesized by debenzylation of compound 1f using hydrogen (60 psi) and 10% Pd/C.Inhibitory activity of diaryl isoxazole derivatives against proliferaction of HCT-116 colorectal cancer cells was evaluated by measurement of the amount of WST-1 formazan formed by adding cell proliferation reagent WST-1. The GI 50 values of these compounds were presented in Table 1. The extent of inhibiting proliferaction of HCT-116 colorectal cancer cells was greater in methoxy group (1a, 1b and 1c) of B-aryl moiety than in hydroxyl group (1d and 1e). The presence of free hydroxyl hydrogen in the B-aryl moiety of KRIBB3 analogues decreased the efficiency of antiproliferative activity. Large benzyloxy substituent (1c...