The dayflower, Commelina communis L., contains 1-deoxynojirimycin (DNJ) and (2R,3R,4R,5R)2,5-bis(hydroxymethyl)-3,4-dihydroxypyrrolidine (DMDP), potent alpha-glucosidase inhibitors. The extracts and powder of this herb are important food materials for prophylaxis against type 2 diabetes. Eleven flavonoid glycosides as antioxidants, isoquercitrin, isorhamnetin-3-O-rutinoside, isorhamnetin-3-O-beta-D-glucoside, glucoluteolin, chrysoriol-7-O-beta-D-glucoside, orientin, vitexin, isoorientin, isovitexin, swertisin, and flavocommelin, were identified from the aerial parts of C. communis. Their antioxidant activities were measured using in vitro assays employing the 1,1-diphenyl-2-picrylhydrazyl radical- and superoxide radical-scavenging assays. The results showed that glucoluteolin, orientin, isoorientin, and isoquercitrin are the predominant antioxidants in this herb. Moreover, isoquercitrin, isorhamnetine-3-O-rutinoside, vitexin, and swertisin inhibited the activity of alpha-glucosidase from rat intestine.
Photoinduced processes of a series of phosphorus tetraphenylporphyrin (PTPP) derivatives ([PTPP-(NHC6H4X)2]+Cl-, X = OCH3, CH3, H, Cl, CF3, and CN) have been investigated by using femtosecond laser flash photolysis mainly. PTPP with OH as an axial ligand showed S2 fluorescence upon excitation of the Soret band. The S2 fluorescence lifetime was estimated to be 1.5 ps. On the other hand, both S2 and S1 fluorescence bands of PTPP-(NHC6H4X)2 were difficult to observe, indicating the existence of an additional deactivation process such as charge separation (CS). From MO calculation and cyclic voltammetry, PTPP and the axial ligand are expected to act as an acceptor and a donor, respectively, upon excitation of PTPP. CS via the S2 state was confirmed during the femtosecond laser flash photolysis by observing the transient absorption of radical anion of PTPP. Furthermore, CS via the S1 state of PTPP was also observed. The CS rate via the S1 state was faster than that from the S2 state. The free energy dependence of the electron-transfer rates was discussed on the basis of Marcus theory.
Alteromonas sp. strain O-7 secretes four chitinases (ChiA, ChiB, ChiC, and ChiD) in the presence of chitin. To elucidate why the strain produces multiple chitinases, we studied the expression levels of the four genes and proteins, their enzymatic properties, and their synergistic effects on chitin degradation. Among the four chitinases, ChiA was produced in the largest quantities, followed by ChiD, and the production of ChiB and ChiC changed at lower levels than those of ChiA and ChiD. The expression of the chiA, chiB, chiC, and chiD genes was investigated at the transcriptional level. The RNA transcript of chiA was most strongly induced in the presence of chitin, the expression of chiD followed, and the RNA transcripts of chiB and chiC changed at low levels. The hydrolyzing activities of the four chitinases against various substrates were examined. ChiA was the most active enzyme against powdered chitin, whereas ChiC was the most active against soluble chitin among the four chitinases. ChiD had activities closer to those of ChiA than to those of ChiB and ChiC. ChiB showed no distinctive feature against the chitinous substrates tested. When powdered chitin was treated with the proper combination of four chitinases, an approximately 2.0-fold increase in the hydrolytic activity was observed. These results, together with the results described above, indicate that ChiA plays a central role in chitin degradation for this strain.Chitin, an insoluble homopolymer of ␤-(1,4)-linked
Alteromonas sp. strain O-7 secretes chitinase A (ChiA), chitinase B (ChiB), and chitinase C (ChiC) in the presence of chitin. A gene cluster involved in the chitinolytic system of the strain was cloned and sequenced upstream of and including the chiA gene. The gene cluster consisted of three different open reading frames organized in the order chiD, cbp1, and chiA. The chiD, cbp1, and chiA genes were closely linked and transcribed in the same direction. Sequence analysis indicated that Cbp1 (475 amino acids) was a chitin-binding protein composed of two discrete functional regions. ChiD (1,037 amino acids) showed sequence similarity to bacterial chitinases classified into family 18 of glycosyl hydrolases. The cbp1 and chiD genes were expressed in Escherichia coli, and the recombinant proteins were purified to homogeneity. The highest binding activities of Cbp1 and ChiD were observed when ␣-chitin was used as a substrate. Cbp1 and ChiD possessed a chitin-binding domain (ChtBD) belonging to ChtBD type 3. ChiD rapidly hydrolyzed chitin oligosaccharides in sizes from trimers to hexamers, but not chitin. However, after prolonged incubation with large amounts of ChiD, the enzyme produced a small amount of (GlcNAc) 2 from chitin. The optimum temperature and pH of ChiD were 50°C and 7.0, respectively.Chitin, an insoluble linear ␤-1,4-linked polymer of N-acetylglucosamine (GlcNAc), is the second most abundant polymer in nature. This polysaccharide is particularly an important nutrient source for maintaining the ecosystem in the marine environment (7). Chitinolytic marine bacteria play a critical role in the process of recycling chitinous materials such as the exoskeletons of crustaceans and insects. If the insoluble form of chitin could not be returned to the ecosystem in a biologically usable form, the marine environment would be completely depleted of a carbon and nitrogen source in a relatively short time (39). To degrade chitin, chitinolytic microorganisms produce two classes of enzymes: chitinases (EC 184.108.40.206) and ␤-N-acetylglucosaminidases (GlcNAcases; EC 220.127.116.11). Chitinases hydrolyze chitin to soluble oligosaccharides, mainly chitobiose, which are further hydrolyzed to GlcNAc by GlcNAcases. Finally, the degradation products, mainly GlcNAc, are then taken up by the cells as a carbon and nitrogen source.Alteromonas sp. strain O-7 is a gram-negative, flagellated, motile, and aerobic rod-shaped bacterium of marine origin (30). This strain was isolated from a sediment sample at Sagami Bay in Japan as an efficient producer of chitinolytic enzymes (30). We have been studying the chitin degradation system of the strain as a model for defining the various components involved in chitin utilization. It was made clear that the chitinolytic system of the strain consists of at least four chitinases (Chi85, ChiA, ChiB, and ChiC) and three ␤-Nacetylglucosaminidases (GlcNAcaseA, GlcNAcaseB, and GlcNAcaseC). The genes for these enzymes have been cloned and characterized previously to clarify the role of individual enzymes in the ...
To date, many antitumor agents have been sythesized or isolated as antibiotics, but all have severe adverse effects due to their strong mammalian toxicities. Therefore, the discovery of less toxic antitumor agents is required. As preliminary step to obtain novel antitumor agents, we have been engaged in searching for cytotoxic activity in natural products. As a result, we have already reported that hinokitiol (b-thujaplicin, Chart 1), 1) the major component of Thujopsis dolabrata SIEB. et ZUCC. var. hondai MAKINO,2) showed in vitro cytotoxic effects on five kinds of human and murine tumor cell lines.3) For example, hinokitiol was found to show an inhibitory effect on a colon 26 cell line with a minimal cytopathogenic concentration of 10 mg/ml. 4) In addition, there are several reports on the inhibitory effects of tropolone as hinokitiol and its derivatives having the same skeleton on tumor cells in vitro.  However, no work has been done on the effects of g-thujaplicin and b-dolabrin, 2) the constituents of the wood of T. dolabrata SIEB. et ZUCC. var. hondai.In this work, as a preliminary step to clarify the antitumor activity of hinokitiol-related compounds, the cytotoxic effects of g-thujaplicin and b-dolabrin (Chart 1), the constituents of T. dolabrata SIEB. et ZUCC. var. hondai on cell lines of human stomach cancer cell KATO-III and Ehrlich's ascites carcinoma in vitro was investigated, in comparison with hinokitiol. As a basic study on the biological activity of hinokitiol-related compounds in mice, acute toxicity following intraperitoneal adminstration of the three compounds was also examined.
MATERIALS AND METHODSChemicals g-Thujaplicin, b-dolabrin and hinokitiol were isolated from acid oil obtained by distillation of the wood of T. dolabrata SIEB. et ZUCC. var. hondai MAKINO according to the method of Nozoe et al., 9) and used for the cytotoxic activity tests. However, as the yield of g-thujaplicin was too small to permit a cytotoxic activity test, a synthetic version was kindly supplied by Takasago Int. Corp. Medium Complete RPMI-1640 medium (Nissui Pharmaceutical Co., Ltd. Japan) containing 100 mg/ml streptomycin and 0.0035 ml/ml 2-mercaptoethanol were used throughout the study. Dulbecco's modified Eagle's medium (DMEM, Nissui Pharmaceutical Co., Ltd. Japan) containing 50 mg/ml kanamycin sulfate and 4mM L-glutamine were used throughout the study.Cells Human stomach cancer cells KATO-III were maintained in DMEM and RPMI-1640 (1 : 1) supplemented by 10% heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY, U.S.A.). Ehrlich's ascites carcinoma cells were maintained in DMEM supplemented by 10% FBS. Each cell line was cultured in each medium at 37°C in a humidified atmosphere of 5% CO 2 and 95% air in a chamber throughout the study.Animals Male Slc: ddY strain mice weighting 26-29 g were used for the acute toxicity test.Cytotoxic Assay Two kinds of tumor cells in the exponential growth phase were plated in 96-well flat-bottom microplates at a density of 3ϫ10 3 cells per 100 ml in eac...
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