Production of luteoskyrin and isolation of a new metabolite, pibasterol, from Penicillium islandicum Sopp.

Production of luteoskyrin, a hepatotoxin synthesized by Penicillium islandicum Sopp., was studied with various fermentation methods. Best results were obtained in static fermentations on glutinous rice at 30 degrees C. The isolated yield of pure luteoskyrin was approximately 400 mg per kg of rice. Also produced were skyrin, islandicin, iridoskyrin, rubroskyrin, chrysophanol, mannitol, and erythritol. A new metabolite, which we call pibasterol, was also isolated.     

Effects of naled and dichlorvos on growth and production of Luteoskyrin byPenicillium islandicum

Two organophosphorus insecticides, 1, 2-dibromo ?2, 2-dichloroethyl dimethyl phosphate (naled) and dimethyl 2, 2 -dichlorovinyl phosphate (dichlorvos) were used for investigation of their effects on growth and production of luteoskyrin mycotoxin byPenicillium islandicum in various cultural media at 25°C for 30 days or 60 days. When the concentration of naled and dichlorvos in Czapek solution broth reached 5mg/50mL, growth and production of luteoskyrin by the fungus was completely inhibited. In unpolished rice medium, 15mg/50g of naled was required to retard fungal growth, while the concentration to inhibit the biosynthesis of luteoskyrin was 10mg/50g. On the other hand, if the medium contained dichlorvos at the level of 30mg/50g, the ability to produce luteoskyrin byP islandicum was significantly reduced. In the unhulled rice case, both naled and dichlorvos at the concentration of 15mg/50g were necessary to retard the fungal growth, and 1 mg/50g of each compound exhibits its ability to inhibit the toxin production. Furthermore, it was also found when the cultural medium contained only small amounts of naled and dichlorvos [0.5mg/50g (mL)] the capability to synthesize luteoskyrin by the fungus was drastically reduced. These data strongly suggest that both naled and dichlorvos have similar ability to inhibit luteoskyrin biosynthesis byP islandicum and are also able to retard the fungal growth.     

Effects of butylated hydroxyanisole,butylated hydroxytoluene and tertiary butylhydroquinone on growth and luteoskyrin production byPenicillium islandicum

Butylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT) and tertiary butylhydroquinone (TBHQ) alone in cultural media were tested for the inhibition of growth and luteoskyrin production by two toxigenic strains ofPenicillium islandicum UST-11 andP. islandicum HLT-6. In potato dextrose agar, the concentrations of BHA and TBHQ from 0.2 mg/disc, BHT from 5.0 mg/disc did affect the growth of both tested strains, but the initial concentrations of these antioxidants to reduced luteoskyrin production by UST-11 strain were BHA 0.5 mg/disc, BHT 1.0 mg/disc and TBHQ 0.4 mg/disc, while for HLT-6, BHA 0.4 mg/disc, BHT and TBHQ were 0.2 mg/disc, respectively. In grainy and powdery rice media, the effects of BHA, BHT and TBHQ on luteoskyrin production byP. islandicum UST-11 and HLT-6 were clearly demonstrated. The efficiency of the inhibitory effect was not only closely related to the concentration of antioxidants, but also completely inhibited the luteoskyrin production at a concentration of 200 mg/kg or higher. Also, the antioxidants at a concentration higher than 20 mg/kg reduced significantly the growth and luteoskyrin production by both strains ofP. islandicum.     

Production of Luteoskyrin, a Hepatotoxic Pigment, by Penicillium islandicum Sopp

Various factors affecting the yields of luteoskyrin, a hepatotoxic mycotoxin, and related pigments in the liquid medium were studied. Maximal yields of luteoskyrin (0.13% by isolation) and of other pigments were attained in the late phase of the cultivation. The yield of the pigment was increased by supplying malt extract, malonic acid, glutamic acid, or asparagine. A useful material for preparation of (14)C-labeled luteoskyrin was 2-(14)C-malonate.     

The interaction of rubroskyrin, a modified bis-anthraquinone pigment fromPenicillium islandicum Sopp, with respiratory chain of liver mitochondria

Summary  Rubroskyrin, a modified bisanthraquinone pigment from an yellow rice moldPenicillium islandicum Sopp, was examined for its redox-interaction with the mitochondrial respiratory chain by using rat liver submitochondrial particles (SMP) and was compared with luteoskyrin and rugulosin. Rubroskyrin showed a redox interaction with the NAD-linked respiratory chain of SMP, promoting NADH oxidase in the presence of rotenone, a specific inhibitor to coupling site I of the respiratory chain. Rubroskyrin-mediated NADH oxidase was not inhibited by antimycin A and cyanide, inhibitors to coupling sites II and III, respectively, indicating a generation of an electron transport shunt from a rotenone-insensitive site of NADH dehydrogenase (complex I) to dissolved oxygen. An electrontransport shunt to cytochromec oxidase from complex I was also observed in the experiment with cytochromec and antimycin A. Rubroskyrin did not interact with succinate-linked respiratory chain. Such enzymatic redox response which generates electron transport shunt was not detected for luteoskyrin and rugulosin in the present study.     

Spin-Trapping and Direct Epr Investigations on the Hepatotoxic and Hepatocarcinogenic Actions of Luteoskyrin, An Anthraquinoid Mycotoxin Produced by Penicillium Islandicum Sopp. Generations of Superoxide Anion and Luteoskyrin Semiquinone Radical in the Redox Systems Consisted of Luteoskyrin and Liver Nadph- Or Nadh-Dependent Reductases

Luteoskyrin is a hepatotoxic and hepatocarcinogenic bisdihydroanthraquinone produced by Penicillium islandicum Sopp. By observing the EPR spectra of DMPO-spin adducts and luteoskyrin semiquinone radical, we investigated in vitro whether luteoskyrin is reduced to its semiquinone radical leading to the generation of active oxygen species in redox systems catalyzed by NADPH-dependent cytochrome reductases of the liver. We found (1) the formation of luteoskyrin semiquinone radical in the NADPH-cytochrome P-450 reductase system under anaerobic conditions, (2) the generation of O- in the systems composed of luteoskyrin, NAD(P)H, and either rat liver microsomal NADPH-cytochrome P-450 reductase or submitochondrial particles and (3) dicoumarol showed no effect on the O- generation in the case of submitochondrial particles. From these results we proposed that luteoskyrin liver injuries are induced by the active oxygen species generated in the process of autoxidation of luteoskyrin semiquinone radical which is produced in the one-electron redox systems catalyzed by the liver NAD(P)H-dependent cytochrome reductases.     

Simple screening method for molds producing intracellular mycotoxins in pure cultures.

A simple screening method for molds producing the intracellular mycotoxins brevianamide A, citreoviridin, cyclopiazonic acid, luteoskyrin, penitrem A, roquefortine C, sterigmatocystin, verruculogen, viomellein, and xanthomegnin was developed. After removing an agar plug from the mold culture, the mycelium on the plug is wetted with a drop of methanol-chloroform (1:2). By this treatment the intracellular mycotoxins are extracted within seconds and transferred directly to a thin-layer chromatography plate by immediately placing the plug on the plate while the mycelium is still wet. After removal of the plug, known thin-layer chromatographic procedures are carried out. The substrate (Czapek yeast autolysate agar) and growth conditions (25 degrees C for 7 days) used by Penicillium taxonomists proved suitable for the production of the mycotoxins investigated when 60 known toxigenic isolates and 865 cultures isolated from foods and feedstuffs were tested with this screening method.     

Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.     

Detection,characterization, and phytotoxic activity of the nucleoside antibiotics,Blasticidin S and 5-Hydroxylmethyl-Blasticidin S

Screening for herbicidal compounds carried out on culture broths of Streptomyces strains isolated from soil resulted in the detection of potent phytotoxic activity. The active principles were isolated, and identified as the nucleoside antibiotics Blasticidin S (Bl-S) and 5-Hydroxymethyl-Blasticidin S (H-M-Bl-S). Bl-S was more active than H-M-Bl-S on seedling germination in petri dishes and in postemergence greenhouse tests. Moreover both antibiotics were more phytotoxic to dicotyledonous than to monocotyledonous plants. The increased sensitivity of dicots was confirmed in carrot and rice cell cultures. Both compounds also inhibited [¹⁴C]amino acid incorporation into proteins of rice and carrot cell cultures. Protein synthesis was more affected in carrot than rice.     

浙江省仓贮稻谷大米真菌区系的研究

在浙江省采集仓贮稻谷及大米样品,共分离到真菌17属48种(不包括酵母菌)。其中:曲霉属(Aspergillus)18种,青霉属(Penicillium)10种,镰孢霉属(Eusarium)4种。研究表明:在浙江省地区之间,贮粮真菌种类无明显差异。稻谷加工成大米后,籽粒表面带菌量及内部带菌率均明显下降,其真菌区系以贮藏真菌为主。稻谷在贮藏前以田间真菌为主。贮藏1—4个月的稻谷因贮藏真菌的种类、数量明显增长,而田间真菌的种类和数量仍保持在较高的水平上,其表面带菌量及谷粒带菌率处于高峰期;贮藏1年以上的稻谷,其主要带菌种类为贮藏真菌,带菌量及带菌率明显下降。早籼谷和晚粳谷在相同贮藏条件下其带菌种类和数量基本一致。     

Nutritional studies of the rice stem borer,Chilo suppressalis Walker,and its mass rearing

The nutritional requirements of the rice stem borer,Chilo suppressalis Walker were studied by rearing on synthetic food media in relation to the constituents of the rice plant. The larvae required amino acids, vitamins, carbohydrates, cholesterol, minerals and water extract of the rice plant for their growth. Among amino acids, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophane and valine were indispensable for the growth, and these amino acids could not be synthesized in the larval body. Among vitamines, thiamine, riboflavin, nicotinic acid, pyridoxine, Ca-pantothenate, folic acid, biotin, cholin chloride and ascorbic acid were essential for the growth. Cholesterol is the only essential nutrient among fat-soluble factors. Fructose, glucose and sucrose showed high nutritive values among carbohydrates. Addition of the water extract of the rice plant to the media improved the development of the insects, but the excess amounts of it resulted inhibition of the growth. Two aromatic acids, benzoic and salicylic acids, were isolated as the growth inhibitory substance. p-methyl acetophenone isolated from the rice plant seemed to play an important role for the attraction of the insect. Mass rearing of the rice stem borer in a laboratory was carried out on two kinds of foods: living rice plant seedlings and synthetic food media. The rearing the insects on rice seedlings is easy if rice seeds are available. The rearing on synthetic food media is modified to simplify from these for the nutritional study. The rearing practices, merit and demerit of both methods are discussed.     

Effects of mycotoxins on the mixed lymphocyte reaction

Effects of toxic fungal metabolites on mixed lymphocyte reaction (MLR) using mouse splenocytes and on growth of mouse myeloma cells were examined. Among 25 toxins assayed, the IC₅₀ values of emodin, luteoskyrin, sterlgmatocystin, deoxynivalenoi, 4-acetylnivalenol, T-2 toxin and fusaric acid for the MLR were lower than those for the cytotoxicity toward the myeloma cells, suggesting that these toxins possess suppressive activity to the cellular immune system.     

黄鳝体表溃疡病病原菌的分离与鉴定

研究首次报道从患体表溃疡病黄鳝的肝脏、肾脏、脾脏等分离到1株细菌, 经动物回归试验鉴定其为病原菌。形态学观察、生理生化试验和16S rRNA基因鉴定, 结果证实该病原菌株为温和气单胞菌(Aeromonas sobria)。通过致病性试验证明其对鲤鱼和泥鳅均有致病力;药敏试验结果表明:分离菌株对头孢氨噻肟、头孢曲松钠、丙氟哌酸、洛美沙星、甲氧苄啶、氟哌酸、壮观霉素等高度敏感,而对氨苄青霉素、乙酰螺旋霉素等药物不敏感。       

cDNA Cloning of Squalene Synthase Genes from Mono- and Dicotyledonous Plants, and Expression of the Gene in Rice

cDNA clones encoding squalene synthases were isolated from rice,maize and soybeans. A phylogenetic tree showed that the enzymesof monocots and dicots form distinct subgroups. In rice, squalenesynthase mRNA was detected in tissues containing dividing cellsand its level was repressed by illumination. (Received July 9, 1997; Accepted October 7, 1997)     

荧光物质(MFA、MFB)的分离、结构鉴定及金华地区高产菌株的筛选

采用硅胶柱层析及制备型液相色谱仪对红曲米中两种荧光物质进行分离纯化,使用高效液相色谱法(HPLC)检测荧光物质纯度,然后使用高分辨质谱(ESI-HRMS)对两种荧光物质进行分析,得到两种荧光物质的分子量分别为356和384,ESI-MS/MS二级质谱把两者鉴定为monasfluore A (MFA)和monasfluore B (MFB);从金华地区红曲米中分离得到10株红曲菌株,经固态发酵采用HPLC法分析,筛选获得1株高产MFA、MFB的菌株WZWZ,该菌株发酵制得红曲米中MFA含量为3.63 g/kg,MFB含量为7.29 g/kg,对WZWZ菌株进行外观形态学及显微观察、ITS基因序列测定与分析,最终将菌株WZWZ鉴定为紫色红曲霉(Monascus purpureus)。     

Mutagenicity of fungal metabolites related to aflatoxin biosynthesis.

Fungal metabolites identified as the intermediates in aflatoxin biosynthetic pathway were screened for their mutagenic activity to Salmonella typhimurium TA98. Norsolorinic acid, averufin, and versiconal acetate were found to possess questionable mutagenic activity, but versicolorin A, and sterigmatocystin were significant mutagens relative to aflatoxin B1. The mutagenic activity appears to be related to the bisfuran and not the anthraquinone moiety of the molecule, even though the latter is a key structure of such potent carcinogenic mycotoxin as luteoskyrin.     

OsCDPK13, a calcium-dependent protein kinase gene from rice,is induced by cold and gibberellin in rice leaf sheath

Calcium-dependent protein kinases (CDPKs) play an important role in rice signal transduction, but the precise role of each individual CDPK is still largely unknown. Recently, a full-length cDNA encoding OsCDPK13 from rice seedling was isolated. To characterize the function of OsCDPK13, its responses to various stresses and hormones were analyzed in this study. OsCDPK13 accumulated in 2-week-old leaf sheath and callus, and became phosphorylated in response to cold and gibberellin (GA). OsCDPK13 gene expression and protein accumulation were up-regulated in response to GA₃ treatment, but suppressed in response to abscisic acid and brassinolide. Antisense OsCDPK13 transgenic rice lines were shorter than the vector control lines, and the expression of OsCDPK13 was lower in dwarf mutants of rice than in wild type. Furthermore, OsCDPK13 gene expression and protein accumulation were enhanced in response to cold, but suppressed under salt and drought stresses. Sense OsCDPK13 transgenic rice lines had higher recovery rates after cold stress than vector control rice. The expression of OsCDPK13 was stronger in cold-tolerant rice varieties than in cold-sensitive ones. The results suggest that OsCDPK13 might be an important signaling component in the response of rice to GA and cold stress.     

Fermentative production of dl-lactic acid from amylase-treated rice and wheat brans hydrolyzate by a novel lactic acid bacterium, Lactobacillus sp.

Rice and wheat brans, without additional nutrients and hydrolyzed by alpha-amylase and amyloglucosidase, were fermented to DL-lactic acid using a newly isolated strain of Lactobacillus sp. RKY2. In batch fermentations at 36 degrees C and pH 6, the amount of lactic acid in fermentation broth reached 129 g l(-1) by supplementation of rice bran with whole rice flour. The maximum productivity was 3.1 g lactic acid l(-1) h(-1) in rice bran medium supplemented with whole rice flour or whole wheat flour.