Continuous alkaline pretreatment of Miscanthus sacchariflorus using a bench-scale single screw reactor.

Miscanthus sacchariflorus 'Goedae-Uksae 1' (GU) was developed as an energy crop of high productivity in Korea. For the practical use of GU for bioethanol production, a bench-scale continuous pretreatment system was developed. The reactor performed screw extrusion, soaking and thermochemical pretreatment at the following operating conditions: 3 mm particle size, 22% moisture content, 140 °C reaction temperature, 8 min residence time, 15 g/min biomass feeding and 120 mL/min NaOH input. As a result of minimizing NaOH concentration and enzyme dosage, 90.8±0.49% glucose yield was obtained from 0.5 M NaOH-pretreated GU containing 3% glucan with 10 FPU cellulase/g cellulose at 50 °C for 72 h. The separate hydrolysis and fermentation of 0.5 M NaOH-pretreated GU containing 10% glucan with 10-30 FPU for 102 h produced 43.0-49.6 g/L bioethanol (theoretical yield, 75.8-87.6%). Thus, this study demonstrated that continuous pretreatment using a single screw reactor is effective for bioethanol production from Miscanthus biomass.

The optimized CO2-added ammonia explosion pretreatment for bioethanol production from rice straw.

A CO2-added ammonia explosion pretreatment was performed for bioethanol production from rice straw. The pretreatment conditions, such as ammonia concentration, CO2 loading level, residence time, and temperature were optimized using response surface methodology. The response for optimization was defined as the glucose conversion rate. The optimized pretreatment conditions resulting in maximal glucose yield (93.6 %) were determined as 14.3 % of ammonia concentration, 2.2 MPa of CO2 loading level, 165.1 °C of temperature, and 69.8 min of residence time. Scanning electron microscopy analysis showed that pretreatment of rice straw strongly increased the surface area and pore size, thus increasing enzymatic accessibility for enzymatic saccharification. Finally, an ethanol yield of 97 % was achieved via simultaneous saccharification and fermentation. Thus, the present study suggests that CO2-added ammonia pretreatment is an appropriate process for bioethanol production from rice straw.

A new cytotoxic spiroketal from the myxobacterium Sorangium cellulosum.

A new spiroketal, spirodienal B (1), was isolated from culture extracts of S. cellulosum. Its structure was elucidated by spectroscopic analysis. In addition to 1, the strain produced a second, closely related antibiotic, spirodienal. Both compounds demonstrated potent cytotoxicity against human tumor cells. Against human tumor cells such as HCT-15, the activity of 1 was more than 4000 times stronger than that of doxorubicin in terms of IC(50).

Soraphinol C, a new free-radical scavenger from Sorangium cellulosum.

A new compound named soraphinol C (1) was isolated from myxobacteria Sorangium cellulosum KM1001 a soil isolate, together with a structurally related known compound, 4-hydroxysattabacin (2). These compounds were isolated by silica gel column chromatography and recycling preparative HPLC, consecutively. The structures of the compounds were determined on the basis of combined spectroscopic analyses. Compounds 1 and 2 exhibited antioxidant activity as a radical scavenger in the experiment using a hydrophilic free-radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride with ORAC values of 0.956 and 0.617, respectively.

Sorangiadenosine, a new sesquiterpene adenoside from the myxobacterium Sorangium cellulosum.

Sorangiadenosine (1), a novel nucleoside possessing a bicyclic, eudesmane-type sesquiterpene as an auxiliary component, was isolated from the culture broth of the gliding bacterium Sorangium cellulosum KM1003, collected from Korean soil. The chemical structure of this compound was determined by combined spectroscopic and chemical analyses. The new compound exhibited moderate antibacterial activity against a wide range of bacterial strains.

Apicularen A induces cell death through Fas ligand up-regulation and microtubule disruption by tubulin down-regulation in HM7 human colon cancer cells.

PURPOSE: Apicularen A has been shown to cause growth inhibition and apoptosis in several cancer cell lines. However, the mechanisms of apicularen A-induced cell death and in vivo effects remain unclear. In this study, we investigated the molecular mechanisms of apicularen A-induced cell death in HM7 human colon cancer cells in vitro and anticancer activity in vivo. EXPERIMENTAL DESIGN: We tested cytotoxicity with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis with DNA fragmentation assay, mitochondrial membrane potential, and cell cycle with fluorescence-activated cell sorting. Caspase activation was done by fluorometry. Alterations of microtubule structure, tubulin protein, and mRNA level were assessed by immunofluorescence, Western blot, and reverse transcription-PCR. In vivo studies were assessed using nude mice tumor cell growth in xenograft model and liver colonization assay. RESULTS: Apicularen A treatment of HM7 cells inhibited cell growth and this inhibition was partially rescued by z-VAD-fmk. Apicularen A caused accumulation of sub-G(1)-G(0), DNA fragmentation, Fas ligand induction, and activation of caspase-8 and caspase-3, but mitochondrial membrane potential was not changed. Furthermore, beta-tubulin protein and mRNA were decreased by apicularen A, but in vitro polymerization of tubulin was not affected. Concurrently, apicularen A-treated cell showed disruption of microtubule architecture. In in vivo studies, apicularen A reduced tumor volume by approximately 72% at the end of a 15-day treatment. Moreover, apicularen A reduced liver colonization as much as 95.6% (50 microg/kg/d). CONCLUSION: Apicularen A induces cell death of HM7 cells through up-regulating Fas ligand and disruption of microtubule architecture with down-regulation of tubulin level. These findings indicate that apicularen A is a promising new microtubule-targeting compound.

Bithiazole metabolites from the myxobacterium Myxococcus fulvus.

Two new bithiazole metabolites (3, 4) along with the previously reported compounds (1, 2) were isolated from the culture broth of the gliding bacterium Myxococcus fulvus collected from a Korean soil. The structures of these compounds were determined to be analogous to myxothiazole A on the basis of combined spectroscopic analyses. The new compounds exhibited significant cytotoxicity and moderate antifungal activity against the mouse fibloblast cell-line L929 and Candida albicans, respectively. These data were consistent with the previous finding that the bioactivity of myxothiazoles was highly dependent on the presence of terminal methoxy enol functionality.

Isolation of tetraprenyltoluquinols from the brown alga Sargassum thunbergii.

Thunbergols A (4) and B (5), tetraprenyltoluquinols, along with three known compounds (1-3) have been isolated from the brown alga Sargassum thunbergii. The structures of these two new compounds were determined to be 9-(3,4-dihydro-2,8-dimethyl-6-hydroxy-2H-1-benzopyran-2-yl)-6-methyl-2-(4-methyl-3-pentenyl)-(2E,6E)-nonadienoic acid (4) and 10-(2,3-dihydro-5-hydroxy-7-methyl-1-benzofuran-2-yl)-10-hydroxy-6-methyl-2-(4-methyl-3-pentenyl)-(2E,6E)-undecadienoic acid (5), respectively, by combined spectroscopic methods. Both of them exhibited significant scavenging activities on radical and potently inhibited generation of ONOO(-) from morpholinosydnonimine (SIN-1).

Inhibition of vacuolar-type (H+)-ATPase by the cytostatic macrolide apicularen A and its role in apicularen A-induced apoptosis in RAW 264.7 cells.

Apicularen A and the known vacuolar-type (H(+))-ATPase (V-ATPase) inhibitor bafilomycin A(1) induced apoptosis of RAW 264.7 cells, while apicularen B, an N-acetyl-glucosamine glycoside of apicularen A, was far less effective. Apicularen A inhibited vital staining with acridine orange of the intracellular organelles of RAW 264.7 cells, inhibited the ATP-dependent proton transport into inside-out microsome vesicles, and inhibited the bafilomycin A(1)-sensitive ATP hydrolysis. The IC(50) values of the proton transport were 0.58 nM for apicularen A, 13 nM for apicularen B, and 0.95 nM for bafilomycin A(1). Furthermore, apicularen A inhibited the bafilomycin A(1)-sensitive ATP hydrolysis more potently than apicularen B. F-ATPase and P-ATPase were not inhibited by apicularen A. We concluded that apicularen A inhibits V-ATPase, and thus induces apoptosis in RAW 264.7 cells.

Induction of nitric oxide production by the cytostatic macrolide apicularen A [2,4-heptadienamide, N-[(1E)-3-[(3S,5R,7R,9S)-3,4,5,6,7,8,9,10-octahydro-7,14 dihydroxy-1-oxo-5,9-epoxy-1H-2-benzoxacyclododecin-3-yl]-1 propenyl]-, (2Z,4Z)-(9CI)] and possible role of nitric oxide in apicularen A-induced apoptosis in RAW 264.7 cells.

We previously reported that apicularen A [2,4-heptadienamide, N-[(1E)-3-[(3S,5R,7R,9S)-3,4,5,6,7,8,9,10-octahydro-7,14 dihydroxy-1-oxo-5,9-epoxy-1H-2-benzoxacyclododecin-3-yl]-1 propenyl]-, (2Z,4Z)-(9CI)], a highly cytostatic macrolide isolated from the myxobacterial genus Chondromyces, induces apoptosis in the mouse leukemic monocyte cell line RAW 264.7. To analyze the action mechanism of apicularen A for the induction of apoptosis, effects of apicularen A on nitric oxide (NO) production in RAW 264.7 cells were examined. It was demonstrated that apicularen A at 10 and 100 nM induced nitrite production, whereas apicularen B [2,4-heptadienamide, N-[(1E)-3-[(3S,5R,7R,9S)-7-[[2-(acetylamino)-2-deoxy-beta-d-glucopyranosyl]oxy]-3,4,5,6,7,8,9,10-octahydro-14-hydroxy-1-oxo-5,9-epoxy-1H-2-benzoxacyclododecin-3-yl]-1 propenyl]-, (2Z,4Z)-(9CI)], an N-acetyl-glucosamine glycoside of apicularen A, had no effect at 100 nM. The apicularen A-induced nitrite production was accompanied by an increase in the level of inducible nitric-oxide synthase (iNOS) and its mRNA and was suppressed by the NOS inhibitor N(G)-monomethyl-l-arginine acetate (l-NMMA). In addition, apicularen A activated nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) and decreased the level of IkappaB-alpha and increased that of phosphorylated c-Jun N-terminal kinase (JNK). Furthermore, the apicularen A-induced nitrite production was suppressed by the NF-kappaB inhibitor Bay 11-7082 [(E)-3-(4-methylphenylsulfonyl)-2-propenenitrile] and the JNK inhibitor SP600125 [anthra[1,9-cd]pyrazol-6(2H)-one]. These findings suggested that apicularen A activates NF-kappaB and AP-1, thus triggering the expression of iNOS mRNA and iNOS protein and induces NO production. Finally, apicularen A decreased cell growth and survival and cell viability and disrupted the mitochondrial membrane potential. The addition of l-NMMA partially recovered the apicularen A-induced decrease in cell growth and survival and cell viability and the disruption of mitochondrial membrane potential. These findings suggested that NO produced by apicularen A treatment participate partially in the apicularen A-induced apoptosis in RAW 264.7 cells.

New macrolides from the sponge Chondrosia corticata.

Three new oxazole-containing metabolites, neohalichondramide (5), (19Z)-halichondramide (6), and secohalichondramide (7), along with four previously reported compounds of the same structural class were isolated from the marine sponge Chondrosia corticata collected from Guam. The structures of novel compounds were determined on the basis of combined spectroscopic analyses. These compounds exhibited significant cytotoxicity and antifungal activity toward the human leukemia cell-line K562 and Candida albicans, respectively.

New sesterterpenes from the sponge Smenospongia sp.

Five new sesterterpenes, three scalarane-type (1-3) and two linear furanosesterterpenes (4, 5), were isolated from the marine sponge Smenospongia sp. collected from Gagu-Do, Korea. The structures of the new compounds, which exhibited moderate cytotoxicity toward the human leukemia cell line K562, were determined from an analysis of spectroscopic data.

New sesterterpenes from the Antarctic sponge Suberites sp.

Suberitenones C and D and suberiphenol, three new sesterterpenes of the suberitane class, were isolated from the sponge Suberites sp. collected from Antarctica. The structures of these compounds were determined on the basis of combined spectral and chemical analyses.

Induction of apoptosis of RAW 264.7 cells by the cytostatic macrolide apicularen A.

In RAW 264.7 cells, a mouse leukaemic monocyte cell line, apicularen A decreased cell growth and survival as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in a concentration-dependent manner at 10-1000 nM. Apicularen B, an N-acetyl-glucosamine glycoside of apicularen A, was 10-100-fold less effective than apicularen A. Apicularen A induced a DNA ladder, an increase in the percentage of sub-G(1) cells and annexin V-binding cells, and promoted the activation of caspase as revealed by the cleavage of poly(ADP-ribose) polymerase, indicating that apicularen A induced apoptosis in RAW 264.7 cells. In addition, apicularen A phosphorylated p44/42 mitogen-activated protein kinase (MAPK) and p38 MAPK. The p44/42 MAPK inhibitor PD98059 rescued the cells from apicularen-induced decrease in cell growth and survival as determined by the MTT assay, while the p38 MAPK inhibitor SB203580 augmented the effect of apicularen A. This suggested the activation of p44/42 MAPK to be pro-apoptotic and the activation of p38 MAPK antiapoptotic in apicularen A-treated RAW 264.7 cells.

Plesiocystis pacifica gen. nov., sp. nov., a marine myxobacterium that contains dihydrogenated menaquinone, isolated from the Pacific coasts of Japan.

Two strains of a novel myxobacterium (designated SIR-1T and SHI-1) were isolated from Japanese coasts located in the Pacific subtropical zone. Cells of both strains were Gram-negative, rod-shaped and motile by gliding. The strains were chemoheterotrophic and strictly aerobic. They had the common characteristics associated with myxobacteria, such as bacteriolytic action and fruiting-body formation. The characteristic feature of the strains was a NaCl growth requirement with an optimum concentration of 2.0-3.0 % (w/v), comparable to that of sea water. In addition, other cationic components of sea water, such as Mg2+, Ca2+ and K+, were needed for growth. The major respiratory quinone was MK-8(H2). The cellular fatty acid profile was characterized by the predominance of iso-C15:0. Characteristic fatty acids anteiso-C16:0 and anteiso-C17:0, and a long-chain polyunsaturated fatty acid (C20:4), were also detected. The G + C content of the genomic DNA of strains SIR-1T and SHI-1 was between 69.3 and 70.0 mol% (as determined by HPLC). Strains SIR-1T and SHI-1 shared almost identical 16S rDNA sequences, and clustered with the genus Nannocystis as their closest relative upon phylogenetic analysis. However, the phylogenetic distance between the novel strains and the genus Nannocystis was large enough to warrant their different generic allocation. This finding was supported by significant phenotypic differences between the novel strains and Nannocystis. Thus, strains SIR-1T and SHI-1 represent a novel genus and species, for which the names Plesiocystis and Plesiocystis pacifica, respectively, are proposed. The type strain of the species is SIR-1T (=JCM 11591T =DSM 14875T =AJ 13960T).

Apicidin, a histone deacetylase inhibitor, induces differentiation of HL-60 cells.

The fungal metabolite apicidin (cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)) inhibited the growth of HL-60 cells in a concentration-dependent manner (100-1000 nM). At higher concentrations (>300 nM), cell death was induced. At 100 nM, it induced hyperacetylation of histone H4 time-dependently, while trichostatin A induced transient hyperacetylation. Apicidin (10-100 nM) increased the cells having nitroblue tetrazolium-reducing activity and expressing CD11b but not CD14 and CD15. The expression of CD11b by apicidin was long lasting, while that by trichostatin A was transient. In K562 cells, apicidin at 10-100 nM did not inhibit cell growth nor express CD11b, CD14 and CD15. Our findings indicate that apicidin inhibits proliferation and induces the early stage of differentiation of HL-60 cells.

Induction of nitric oxide synthase by Oldenlandia diffusa in mouse peritoneal macrophages.

Oldenlandia diffusa (OD) has been used to treat malignant tumors. In this study using mouse peritoneal macrophages, we have examined the mechanism by which OD regulates nitric oxide (NO) production. When OD (1 mg/ml) was used in combination with 10 U/ml of recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production (36.13+/-7.12 microM) by the Griess method (nitrite). Treatment of macrophages with rIFN-gamma plus OD (1 mg/ml) caused a significant increase in tumor necrosis factor-alpha (TNF-alpha) production (4.49+/-1.43 ng/ml) by enzyme-linked immunosorbent assay. The increased production of NO and TNF-alpha from rIFN-gamma-plus OD-stimulated cells was almost completely inhibited by pretreatment with 100 microM of pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kappaB). PDTC also inhibited phosphorylation of IkappaB in rIFN-gamma-plus OD-stimulated cells. These findings demonstrate that OD increases the production of NO and TNF-alpha by rIFN-gamma-primed macrophages and suggest that NF-kappaB plays a critical role in mediating these effects of OD.

Inhibitory effect of apoptosis in human astrocytes CCF-STTG1 cells by lemon oil.

The effects of lemon pure essential oils on the heat shock-induced apoptosis in human astrocytes cell line CCF-STTG1 were examined. In previous studies, heat shock has been reported to induce the apoptosis or programmed cell death through the activation of caspase-3. Treatment of heat shock on CCF-STTG1 cells markedly induced apoptotic cell death as determined by flow cytometry. Interestingly, pre-treatment with lemon pure essential oils on CCF-STTG1 cells inhibited the heat shock-induced apoptosis. Lemon oil also inhibited the heat shock-induced apoptosis in primary cultured rat astrocytes. To determine whether lemon oil inhibits the heat shock-induced activation of the apoptotic proteases, activation of caspase-3 was assessed by Western blotting. DNA fragmentation, giemsa staining, and heat shock-induced activation of caspase-3 were blocked by lemon pure essential oil, which is consistent with flow cytometry. Poly-ADP-ribose polymerase (PARP), the cysteine protease substrate, was fragmented as a consequence of apoptosis by heat shock. Lemon oil inhibited the PARP fragmentation. These results suggest that lemon pure essential oils may modulate the apoptosis through the activation of the interleukin-1 beta -converting enzyme-like caspases.

ATF is important to late S phase-dependent regulation of DNA topoisomerase IIalpha gene expression in HeLa cells.

DNA topoisomerase IIalpha (Topo IIalpha) is regulated in late S phase-dependent manner. To identify late S phase-dependent cis-acting elements of Topo IIalpha gene, we have investigated the synchronized HeLa cells with chloramphenicol acetyltransferase and DNase I footprinting assays. The level of Topo IIalpha mRNA increased after release from aphidicolin block and reached a maximum in 8h (late S phase) in HeLa cells, and Topo II unknotting activity was also in parallel with the level of Topo IIalpha mRNA. The late S phase-regulatory element was found to be located in the region containing ATF-binding element between -290 and -90bp and the region was required for a maximal stimulation during late S phase. DNase I footprinting assay showed that ATF-binding element and novel cis-acting element (Topo IIalpha-specific sequence) were the principal protein-binding sites and the proteins interacting with these elements were induced during late S phase. One DNA-protein complex was formed by DNA mobility shift assay when ATF-binding site was incubated with nuclear extract prepared from late S phase cells, but no protein bound in non-S phase cells. Taken together, these results suggest that ATF may be essential transacting factor for maximal expression of Topo IIalpha gene during late S phase in HeLa cells.

Hydrazinocurcumin, a novel synthetic curcumin derivative, is a potent inhibitor of endothelial cell proliferation.

Curcumin and some of its derivatives were known as in vivo inhibitors of angiogenesis. In present study, a novel curcumin derivative, named hydrazinocurcumin (HC) was synthesized and examined for its biological activities. HC potently inhibited the proliferation of bovine aortic endothelial cells (BAECs) at a nanomolar concentration (IC(50)=520 nM) without cytotoxicity. In vivo and in vitro angiogenesis experiments showed HC as a new candidate for anti-angiogenic agent.