Isolation of a novel deep-sea Bacillus circulus strain and uniform design for optimization of its anti-aflatoxigenic bioactive metabolites production.

The deep-sea bacterium strain FA13 was isolated from the sediment of the South Atlantic Ocean and identified as Bacillus circulans based on 16S ribosomal DNA sequence. Through liquid fermentation with five media, the cell-free supernatant fermented with ISP2 showed the highest inhibition activities against mycelial growth of Aspergillus parasiticus mutant strain NFRI-95 and accumulation of norsolorinic acid, a precursor for aflatoxin production. Based on ISP2, uniform design was used to optimize medium formula and fermentation conditions. After optimization, the inhibition efficacy of the 20-time diluted supernatant against A. parasiticus NFRI-95 mycelial growth and aflatoxin production was increased from 0-23.1% to 100%. Moreover, compared to the original protocol, medium cost and fermentation temperature were significantly reduced, and dependence on seawater was completely relieved, thus preventing the fermentor from corrosion. This is the first report of a deep-sea microorganism which can inhibit A. parasiticus NFRI-95 mycelial growth and aflatoxin production.

Synthetic RNA Silencing of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2).

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.

Identified biosynthetic pathway of aspergiolide A and a novel strategy to increase its production in a marine-derived fungus Aspergillus glaucus by feeding of biosynthetic precursors and inhibitors simultaneously.

Aspergiolide A is a novel anti-tumor anthraquinone derivant produced by marine-derived fungus Aspergillus glaucus. To identify its biosynthetic pathway and further improve the production, the effects of biosynthetic pathway specific inhibitors and precursors were investigated. Cerulenin and iodoacetamide, the specific inhibitors of polyketide pathway, could completely inhibit the aspergiolide A accumulation. Putative precursors of polyketide pathway could increase aspergiolide A production greatly, such as 6 mM acetate increased production by 135%. Simvastatin and citrate, the inhibitors of mevalonate pathway, stimulated the production by 63% and 179%, respectively. Considering that acetyl-CoA is the common starter unit in both polyketide and mevalonate pathway, a novel strategy was designed to stimulate the aspergiolide A accumulation. Combinations of 12 mM acetate with 0.3 mM simvastatin could increase the production by 151%, while the supplementation with 12 mM acetate and 12 mM citrate brought a 262% increase of aspergiolide A production. The strategy might be very useful to enhance the production of other secondary metabolites derived from polyketide pathway.

Combined experimental and theoretical study on photoinduced toxicity of an anthraquinone dye intermediate to Daphnia magna.

The toxicity of chemicals can be enhanced by light through two photochemical pathways: Photomodification to more toxic substances and photosensitization. In the present study, the reactive oxygen species (ROS) mechanism for photoinduced acute toxicity of 1-amino-2,4-dibromoanthraquinone (ADBAQ) to Daphnia magna was clarified by experiment and theoretical calculation. The results of the present study show that ADBAQ exhibited high toxicity to D. magna under simulated solar radiation (SSR), with a median effective concentration of 1.23 +/- 0.19 nM (mean +/- standard deviation). The photomodified ADBAQ (mixtures of ADBAQ and its photoproducts) was less phototoxic than the intact ADBAQ. The SSR-only or ADBAQ-only treatments did not affect the ROS level in D. magna, whereas increased ROS levels were observed in the presence of SSR and ADBAQ. The ROS in vivo were determined by measuring the fluorescence of 2',7'-dichlorofluorescein, which is a useful technique to assess toxicity of chemicals to aquatic organisms. The antioxidants, including vitamin C, vitamin E, and beta-carotene, decreased the photoinduced oxidative damage to D. magna, probably by scavenging ROS. These experimental results demonstrate that photosensitization is the potential mechanism of photoinduced toxicity of ADBAQ to D. magna. Proposed phototoxic pathways of ADBAQ were elucidated by means of time-dependent density functional theory. The theoretical calculation indicates that superoxide anion and singlet oxygen are able to be generated through electron transfer or energy transfer in the photosensitization reactions.

Quinone-respiration improves dechlorination of carbon tetrachloride by anaerobic sludge.

The impact of humic acids and the humic model compound, anthraquinone-2,6-disulfonate (AQDS), on the biodegradation of carbon tetrachloride (CT) by anaerobic granular sludge was studied. Addition of both humic acids and AQDS at sub-stoichiometric levels increased the first-order rate of conversion of CT up to 6-fold, leading to an increased production of inorganic chloride, which accounted for 40-50% of the CT initially added. Considerably less dechlorination occurred in sludge incubations lacking humic substances. By comparison, very limited dechlorination occurred in sterile controls with autoclaved sludge. Accumulation of chloroform (1-10%) and dichloromethane (traces) also accounted for the CT converted. The accumulation of a chlorinated ethene, perchloroethylene (up to 9% of added CT), is also reported for the first time as an end-product of CT degradation. A humus-respiring enrichment culture (composed primarily of a Geobacter sp.) derived from the granular sludge also dechlorinated CT, yielding products similar to the AQDS-supplemented granular sludge consortium. The dechlorination of CT by the Geobacter enrichment was dependent on the presence of AQDS or humic acids, which were reduced during the assays. The reduced form of AQDS, anthrahydroquinone-2,6-disulfonate, was shown to cause the chemical reduction of CT when incubated in sterile medium. The results taken as a whole indicate that the formation of reduced humic substances by quinone-respiring microorganisms can contribute to the reductive dechlorination of CT.

Induction of apoptosis in human cancer cell lines by the novel anthracenyl-amino acid topoisomerase I inhibitor NU/ICRF 505.

Anthracenyl-amino acid conjugates represent a novel chemical class of topoisomerase (topo) inhibitor. NU/ICRF 505 is a lead compound that stabilises topo I cleavable complexes and is actively cytotoxic at low microM concentrations. In this study, endonucleolytic DNA cleavage was used as a marker of apoptosis to investigate mechanisms of cell death produced by this compound. NU/ICRF 505 (5 microM) induced a substantial increase in the level of DNA fragmentation in HL60 cells (up to 30% of total extracted DNA) but only after a 48 and 72 h drug exposure (compared with 6 h after treatment with camptothecin), as determined qualitatively by conventional gel electrophoresis and quantitatively by spectrofluorimetry. This effect was substantially reversed by co-treatment with zinc (1 mM). Subsequent studies with the human lung (NX002), ovarian (A2780) and colon (HT29) cancer cell lines yielded evidence of formation of higher molecular weight DNA fragments in NX002 and A2780 cells in response to NU/ICRF 505 (5 microM). Co-treatment with zinc (1 mM) caused a small decrease in DNA fragmentation. These data suggest that the induction of apoptosis may play an important role in the mechanism of cytotoxicity of NU/ICRF 505 in HL60 cells and that other pathways of cell death may also be operative in NX002 and A2780 in conjunction with apoptosis.

Suppression of the purgative action of rhein anthrone, the active metabolite of sennosides A and B, by calcium channel blockers, calmodulin antagonists and indomethacin.

The involvement of Ca2+ in the mechanism of the purgative action of rhein anthrone was studied. Among individual or combination pretreatments with calcium channel blockers, calmodulin antagonists and prostaglandin biosynthesis inhibitors, the combination of indomethacin and nifedipine completely blocked the diarrhoea induced by rhein anthrone and also inhibited its effects on colonic fluid and electrolyte transport, and large intestinal motility. Calmodulin antagonists were less active regarding suppression of the effects of rhein anthrone. We concluded that, in addition to prostaglandins, diarrhoea induced by rhein anthrone must also involve the calcium channel which can be blocked by nifedipine, but not verapamil.

Serotonin antagonists inhibit sennoside-induced fluid secretion and diarrhea.

The aim of this study was to investigate whether 5-hydroxytryptamine (serotonin, 5-HT) is involved in the mediation of sennoside-induced colonic fluid secretion and diarrhea. Oral administration of purified sennosides (25, 40 and 64 mg/kg) dose-dependently reversed net fluid absorption to net fluid secretion, enhanced the incidence of diarrhea and stimulated the release of 5-HT into the colonic lumen from 7.1 to 17.3 ng/g wet weight. The 5-HT2 antagonist ketanserin and the 5-HT3 antagonist tropisetron dose-dependently but only partially reduced sennoside (40 mg/kg)-induced fluid secretion whereas the 5-HT3 antagonist granisetron dose-dependently reduced and at 300 micrograms/kg totally abolished sennoside-induced secretion. Granisetron, but not ketanserin and tropisetron, reduced the incidence of diarrhea in sennoside-treated rats, indicating the involvement of 5-HT also in acceleration of large intestinal transit. It is concluded that 5-HT is an important mediator both of sennoside-induced fluid secretion in the rat colon and of diarrhea.

Rhein stimulates electrogenic chloride secretion by activation of submucosal neurons in guinea pig colon.

Conventional flux chamber methods were applied to investigate the mode of action of rhein, an active metabolite derived from colonic microbial fermentation of the naturally occurring sennoside laxatives, in muscle-stripped segments of guinea pig colon. Mucosal or serosal application of rhein (10 nmol/1 to 0.5 mmol/l) resulted in a dose-dependent increase in short-circuit current (Isc) that was superimposed by irregular fluctuations in Isc. The response to electrical field stimulation was increased. The rhein-evoked increase in Isc was reduced by serosal addition of 50 mumol/l bumetanide, 1 mumol/l tetrodotoxin, 1 mumol/l atropine and 10 mumol/l piroxicam but not 100 mumol/l hexamethonium, 1 mumol/l ICS 205 930 or 10 mumol/l cimetidine. The study suggests that rhein activates chloride secretion by excitation of submucosal neurons and release of acetylcholine and endogenous prostaglandins, but not by release of histamine or serotonin.

Inhibitory effect of the non-steroidal anti-inflammatory drug, indomethacin on the naturally occurring carcinogen, 1-hydroxyanthraquinone in male ACI/N rats.

The effect of the non-steroidal anti-inflammatory drug indomethacin on 1-hydroxyanthraquinone (1-HA)-induced carcinogenesis was investigated in male ACI/N rats. Animals were fed the diet containing 1.5% 1-HA and simultaneously given indomethacin solution (16 p.p.m.) as drinking water for 48 weeks. The incidences of large bowel neoplasms (adenomas and adenocarcinomas) and forestomach (papillomas) in rats given 1-HA and indomethacin (large intestinal tumors: 0/14, 0%; forestomach tumors: 2/14, 14%) were significantly lower than those in rats given 1-HA alone (large intestinal tumors: 12/27, 44%; forestomach tumors: 14/27, 52%) (P = 0.002 and P = 0.01 respectively). Liver cell adenomas were developed in a rat given 1-HA and no liver tumors in rats treated with 1-HA and indomethacin. Altered liver cell foci were present in rats given 1-HA alone (18/27, 67%) and those given 1-HA and indomethacin (8/14, 57%), but no significant difference in the incidence between the two groups was found. Untreated animals and rats given indomethacin alone had no neoplasms in the large bowel, forestomach and liver. Thus, the non-steroidal anti-inflammatory drug indomethacin significantly inhibited carcinogenesis induced by the naturally occurring carcinogen, 1-HA.

Suppression of the purgative action of rhein anthrone, the active metabolite of sennosides A and B, by indomethacin in rats.

Rhein anthrone (12.48 mg kg-1) produces watery and mucoid diarrhoea approximately 20 min after intracaecal administration to rats. Pretreatment with the prostaglandin (PG) biosynthesis inhibitor indomethacin (10 mg kg-1, i.p.) only delayed and did not completely block the onset of the induced diarrhoea. Rhein anthrone stimulated PGE2 release into the rat colonic lumen and the increased release was depressed by indomethacin. Rhein anthrone also accelerated large intestinal transit and this acceleration could be partly inhibited by indomethacin, which was probably responsible for the delay in the onset of diarrhoea. Indomethacin prevented the enhanced water, K+ and mucus secretion and the reduced Na+ absorption in the colon which were induced by rhein anthrone. The net water secretion could not be reversed to net absorption and the mucus secretion was only slightly depressed by indomethacin. Thus, our findings suggest that other mechanisms, together with the PG-dependent mechanism, are involved in the purgative action of rhein anthrone in rats.

Structure-activity relationships (SAR) of hydroxyapatite-binding molecules.

Carboxyl-containing molecules can bind to HA with sufficient affinity to prevent the uptake of AZ by this mineral. Structural features that favor binding can be discerned. Isolated carboxyls are inactive; adjacent dicarboxylic functions are marginally active. Crowding of oxygens in the form of vicinal carboxyls or hydroxyls contributes the property of HA binding ability. This property exhibits gradation in magnitude and the variation can be correlated with structural features of the carboxyl-containing molecule. Phospho functions are generally more powerful contributors to HA binding property than are carboxyls.

The antagonistic effect of morphine on rhein-stimulated fluid, electrolyte and glucose movements in guinea-pig perfused colon.

Rhein (1,8-dihydroxy-3-carboxyanthraquinone), in a concentration of 6 X 10(-4)M, inhibits water absorption from the colon and causes a net transfer of fluid and electrolyte into the intestinal lumen. Morphine (4 X 10(-4)M) counteracted the water and electrolyte secretion. Prior perfusion with morphine protected the large intestine from the laxative effect of a rhein perfusion. Differences in absorption rate of 99mTc-EDTA, a poorly absorbable marker, were found, as morphine caused nearly all radioactive compound to be retained in the colon, while rhein significantly facilitated the transfer of marker from colon through mucosal barrier to blood. The route followed by the 99mTc-EDTA complex was not the same as that followed by water, suggesting that 99mTc-EDTA travels by a paracellular route. Morphine counteracted the inhibition of Na+ absorption caused by rhein and antagonized the massive loss of K+ incurred by the presence of rhein in the colon. Cl- absorption is reversed to secretion in the presence of rhein while normal values were restored by morphine. Neither the HCO-3 content nor the pH were affected by either drug. Active absorption of glucose was completely blocked in the presence of rhein; the block could be antagonized by morphine.

Stimulation of PGE2 synthesis and water and electrolyte secretion by senna anthraquinones is inhibited by indomethacin.

The effect of dried senna pod extract, containing 10% sennoside B, on colonic electrolyte and fluid transport was examined in the anaesthetized rat in-situ. Oral administration of senna pod extract dose-dependently (17.5-30 mg kg-1, calculated as sennoside B) reversed net absorption of water, sodium and chloride to net secretion and increased potassium secretion. Senna pod extract stimulated the output of prostaglandin E2 into the colonic lumen. Inhibition of prostaglandin biosynthesis by pretreatment of the rats with indomethacin (10 mg kg-1) significantly inhibited the effects of senna pod extract (17.5-30 mg kg-1) both on net fluid transport and on prostaglandin E2 synthesis. The inhibitory effect of indomethacin on net fluid transport induced by senna pod extract (30 mg kg-1) was dose-dependent. It is concluded that anthraquinones exert their laxative action at least partially via stimulation of colonic fluid and electrolyte secretion, and that this secretion is mediated by stimulation of endogenous prostaglandin E2 formation.

Protection by testosterone propionate against the nephrotoxicity of 2-aminoanthraquinone in Fischer rats.

The role of gonadal hormones in the nephrotoxicity of the hepatocarcinogen 2-aminoanthraquinone (2-AAQ) was investigated. Intact and castrated four week old Fischer rats of both sexes and castrated female rats with subcutaneous implants of testosterone propionate pellets were fed 2% 2-AAQ in a Wayne meal diet. After 12 weeks of ad libitum feeding the animals were then given control diet. Based on survival, body, liver and kidney weights, and the histopathological evaluation of the kidneys, castrated female rats given testosterone propionate were afforded the greatest protective effect against the nephrotoxicity of 2-AAQ.