Fumonisins--novel mycotoxins with cancer-promoting activity produced by Fusarium moniliforme

Cultures on corn of Fusarium moniliforme MRC 826 are known to cause leukoencephalomalacia in horses and to be toxic and hepatocarcinogenic in rats. Culture material of this F. moniliforme isolate has also been shown to exhibit cancer-promoting activity in a short-term cancer initiation-promotion bioassay with diethylnitrosamine-initiated rats and the induction of gamma-glutamyl-transpeptidase-positive (GGT+) foci as an endpoint after 4 weeks of promotion. This bioassay was used as a monitoring system to isolate cancer-promoting compounds from cultures of F. moniliforme MRC 826. Culture material was successively extracted with ethyl acetate and CH3OH-H2O (3:1). Most of the cancer-promoting activity was recovered in the CH3OH-H2O extract and remained in the aqueous phase following partitioning of this extract between CH3OH-H2O (1:3) and CHCl3. The CH3OH-H2O fraction was chromatographed on an Amberlite XAD-2 column, and the active fraction was eluted with CH3OH. This fraction was chromatographed on a silica gel column with CHCl3-CH3OH-CH3COOH (6:3:1) as eluent and further purified on a C18 reverse-phase column. Two pure compounds were isolated, and these have been chemically characterized and given the trivial names fumonisin B1 and B2. At least 2 g of the major compound fumonisin B1 was purified from 1 kg of culture material. Fumonisin B1 in the diet (0.1%) significantly (P less than 0.001) induced the formation of GGT+ foci in the livers of initiated as well as noninitiated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survey of fumonisin production by Fusarium species

Fumonisins B1 (FB1) and B2 (FB2), two structurally related mycotoxins with cancer-promoting activity, were recently isolated from corn cultures of Fusarium moniliforme MRC 826. These toxins have been reported to be produced also by isolates of F. proliferatum. Contamination of foods and feeds by F. moniliforme has been associated with human esophageal cancer risk, and FB1 has been shown to be the causative agent of the neurotoxic disease leukoencephalomalacia in horses. Because of the toxicological importance of the fumonisins, the potential to produce FB1 and FB2 was determined in a study of 40 toxic Fusarium isolates representing 27 taxa in 9 of the 12 sections of Fusarium, as well as two recently described species not yet classified into sections. With the exception of one isolate of F. nygamai, fumonisin production was restricted to isolates of F. moniliforme and F. proliferatum, in the section Liseola. The F. nygamai isolate produced 605 micrograms of FB1 g-1 and 530 micrograms of FB2 g-1, and the identity of the toxins was confirmed by capillary gas chromatography-mass spectrometry. This is the first report of the production of the fumonisins by F. nygamai.     

Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme.

The kinetics of the production of fumonisin B1 (FB1) by Fusarium moniliforme MRC 826 in corn cultures was investigated as a function of fungal growth at various incubation temperatures. The growth rate of F. moniliforme, as measured by ergosterol concentration, was higher at 25 degrees C than at 20 degrees C, reaching a stationary phase after 4 to 6 weeks in both cases. FB1 production commenced after 2 weeks during the active growth phase, continued to increase during the stationary phase, and decreased after 13 weeks. The overall maximal yield of FB1 (17.9 g/kg, dry weight) was obtained in corn cultures incubated at 20 degrees C for 13 weeks, but it was not significantly (P greater than 0.05) higher than the maximum yield (16.5 g/kg, dry weight) obtained at 25 degrees C after 11 weeks. However, a significantly (P less than 0.05) higher mean yield was detected at 25 degrees C (9.5 g/kg, dry weight) than at 20 degrees C (8.7 g/kg, dry weight). Production reached a plateau after 7 weeks of incubation at 25 degrees C or 9 weeks of incubation at 20 degrees C. The maximal production of FB1 at 30 degrees C was very low (0.6 g/kg, dry weight). FB1 was also found to be heat stable, as there was no reduction in the FB1 concentration after boiling culture material of F. moniliforme MRC 826.     

Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme

The kinetics of the production of fumonisin B1 (FB1) by Fusarium moniliforme MRC 826 in corn cultures was investigated as a function of fungal growth at various incubation temperatures. The growth rate of F. moniliforme, as measured by ergosterol concentration, was higher at 25 degrees C than at 20 degrees C, reaching a stationary phase after 4 to 6 weeks in both cases. FB1 production commenced after 2 weeks during the active growth phase, continued to increase during the stationary phase, and decreased after 13 weeks. The overall maximal yield of FB1 (17.9 g/kg, dry weight) was obtained in corn cultures incubated at 20 degrees C for 13 weeks, but it was not significantly (P greater than 0.05) higher than the maximum yield (16.5 g/kg, dry weight) obtained at 25 degrees C after 11 weeks. However, a significantly (P less than 0.05) higher mean yield was detected at 25 degrees C (9.5 g/kg, dry weight) than at 20 degrees C (8.7 g/kg, dry weight). Production reached a plateau after 7 weeks of incubation at 25 degrees C or 9 weeks of incubation at 20 degrees C. The maximal production of FB1 at 30 degrees C was very low (0.6 g/kg, dry weight). FB1 was also found to be heat stable, as there was no reduction in the FB1 concentration after boiling culture material of F. moniliforme MRC 826.     

Production of [14C]fumonisin B1 by Fusarium moniliforme MRC 826 in corn cultures.

Kinetics of growth and fumonisin production by Fusarium moniliforme MRC 826 in corn "patty" cultures were investigated, and a technique was developed for the production of [14C]fumonisin B1 ([14C]FB1) by using L-[methyl-14C]methionine as the precursor. A significant (P < 0.01) correlation exists between fungal growth and FB1 (r = 0.89) and FB2 (r = 0.87) production in corn patties, beginning after 2 days and reaching the stationary phase after 14 days of incubation. [14C]FB1 was produced by adding L-[methyl-14C]methionine daily to cultures during the logarithmic phase of production. Incorporation of the isotope occurred at C-21 and C-22 of the fumonism molecule and was enhanced in the presence of unlabeled L-methionine. Although the concentration of exogenous unlabeled methionine is critical for incorporation of the 14C label, optimum incorporation was achieved by adding 50 mg of unlabeled L-methionine and 200 mu Ci of L-[methyl-14C]methionine to a corn patty (30 g) over a period of 9 days, yielding [14C]FB1 with a specific activity of 36 mu Ci/mmol.     

Fumonisins: Isolation,chemical characterization and biological effects

The fumonisin B mycotoxins (FB₁ and FB₂) have been purified and characterized from corn cultures of Fusarium moniliforme strain MRC 826. Fumonisin B₁ (FB₁, the major fumonisin produced in culture, has been shown to be responsible for the major toxicological effects of the fungus in rats, horses and pigs. Recent investigations on the purification of compounds with chromatographic characteristics similar to FB₁ have led to the identification of two new fumonisins, FB₃ and FB₄. Fumonisins A₁ and A₂, the N-acetyl derivatives of FB₁ and FB₂ respectively, were also purified and shown to be secondary metabolites of the fungus. Short-term carcinogenesis studies in a rat liver bioassay indicated that over a period of 15 to 20 days, at dietary levels of 0.05–0.1%, FB₂ and FB₃ closely mimic the toxicological and cancer initiating activity of FB₁ and thus could contribute to the toxicological effects of the fungus in animals. In contrast, no biological activity could be detected for FA₁ under identical experimental conditions. These studies and others have indicated that the fumonisin B mycotoxins, although lacking mutagenicity in the Salmonella test or genotoxicity in the DNA repair assays in primary hepatocytes, appear to induce resistant hepatocytes similar to many known hepatocarcinogens.     

5—氟尿苷的微生物转化

5 氟尿苷 (简称ＦＵＲ)是抗肿瘤核苷药物脱氧氟尿苷 (Ｆｌｏｘｕｒｉｄｉｎｅ ,简称ＤＦＵＲ)的合成中间体。脱氧氟尿苷是一种抗代谢类抗肿瘤药 ,在体内可以部分转化为氟尿嘧啶 (简称ＦＵ) ,二者具有相似的作用途径和抗肿瘤谱。与ＦＵ相比 ,由于ＤＦＵＲ的抗肿瘤活性高且毒副反应小 ,主要用于治疗晚期结直肠癌和各种类型肝癌。在国内 ,采用化学法合成的ＤＦＵＲ业已进入临床研究阶段[1]采用化学合成法生产ＤＦＵＲ时 ,由于反应过程中需将碱基或核糖残基的部分基团进行保护 ,而且产物为多种核苷异构体和其它副产品的混合物 ,需要进一步分离 ,…     

A new fumonisin from solid cultures of Fusarium moniliforme

A new fumonisin has been isolated from Fusarium moniliforme isolate MRC826 grown on corn. It was shown by NMR and mass spectrometry to be an isomer of fumonisin B₂ that has free hydroxyl groups at C-3 and C-10 instead of the normal C-3 and C-5. This new fumonisin was detected in cultures of most isolates of F. moniliforme that were examined and was usually present at concentrations similar to those of fumonisin B₂. Two isolates of F. moniliforme that produce significantly higher levels of this new isomer were identified.Abbreviations ELEM equine leukoencephalomalacia Mention of companies or products by name does not imply their endorsement by the US Department of Agriculture over others not cited.     

Separation of Brain Dolichol Kinase from Endogenous Activating Factors: Evidence That Phospholipid Enhances the Interaction Between Enzyme and Dolichol

Porcine brain dolichol kinase activity is effectively solubilized by extracting salt-washed microsomes with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). When the detergent-solubilized activity is chromatographed on Sepharose CL-6B, a low amount of dolichol kinase activity is recovered in the void volume, and a dolichol kinase activator (DKA) is eluted (Ve/Vo = 1.9-2.2) with the bulk of the membrane phospholipids. Although only approximately 20% of the activity applied to the Sepharose CL-6B column is detected in the column fractions, virtually all of the original activity is restored when the Vo fraction is recombined with DKA. Endogenous DKA, isolated from brain microsomes, is heat-stable, is extractable with CHCl3/CH3OH (2:1), and has the chemical and chromatographic properties of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Moreover, approximately 50% of the stimulatory activity is lost when the PC present in the DKA fraction is degraded by purified phospholipase C from Clostridium perfringens. Also consistent with a phospholipid co-factor requirement, the dolichol kinase activity recovered in the partially phospholipid-depleted fraction (Vo) is markedly stimulated by various molecular species of exogenous purified PC or PE, but not by phosphatidylinositol, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, or sphingomyelin. A comparison of defined molecular species shows that PCs containing oleoyl or linoleoyl groups in the 1 and 2 positions are the most stimulatory, suggesting that the fatty acyl moieties are involved in the enzyme-phospholipid interaction. Kinetic analyses indicate that PC enhances the interaction between dolichol kinase and dolichol, the lipophilic substrate, but does not alter the apparent Km for CTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme

Culture materials and grains contaminated with certain isolates of Fusarium moniliforme cause equine leucoencephalomalacia, porcine pulmonary edema syndrome, and liver cancer in rats. The causative agents are thought to be a family of compounds called fumonisins, which bear considerable structural similarity to the long-chain (sphingoid) base backbones of sphingolipids. Incubation of rat hepatocytes with fumonisins inhibited incorporation of [14C]serine into the sphingosine moiety of cellular sphingolipids with an IC50 of 0.1 microM for fumonisin B1. In contrast, fumonisin B1 increased the amount of the biosynthetic intermediate sphinganine, which suggests that fumonisins inhibit the conversion of [14C]sphinganine to N-acyl-[14C]sphinganines, a step that is thought to precede introduction of the 4,5-trans double bond of sphingosine (Merrill, A.H., Jr. and Wang, E. (1986) J. Biol. Chem. 261, 3764-3769). In agreement with this mechanism, fumonisin B1 inhibited the activity of sphingosine N-acyltransferase (ceramide synthase) in rat liver microsomes with 50% inhibition at approximately 0.1 microM and reduced the conversion of [3H]sphingosine to [3H]ceramide by intact hepatocytes. As far as we are aware, this is the first discovery of a naturally occurring inhibitor of this step of sphingolipid metabolism. These findings suggest that disruption of the de novo pathway of sphingolipid biosynthesis may be a critical event in the diseases that have been associated with consumption of fumonisins.     

Influence of kernel age on fumonisin B1 production in maize by Fusarium moniliforme.

Production of fumonisins by Fusarium moniliforme on naturally infected maize ears is an important food safety concern due to the toxic nature of this class of mycotoxins. Assessing the potential risk of fumonisin production in developing maize ears prior to harvest requires an understanding of the regulation of toxin biosynthesis during kernel maturation. We investigated the developmental-stage-dependent relationship between maize kernels and fumonisin B1 production by using kernels collected at the blister (R2), milk (R3), dough (R4), and dent (R5) stages following inoculation in culture at their respective field moisture contents with F. moniliforme. Highly significant differences (P 相似文献   

Production of fumonisins by Fusarium moniliforme strains isolated from corn grain

Fusarium moniliforme is the predominant fusarium species in the grain mycoflora of corn grown in Northern Caucasus, accounting for 95% of fusarium isolates. Eighty-five Fusarium moniliforme strains were grown on grain substrate and checked for the presence of fumonisins (B1 + B2 + B3) by indirect solid-phase enzyme immunoassay (EIA). All strains were capable of producing fumonisins (0.95 to 32,000 mg/kg). Strains sampled in the Krasnodar krai produced the highest fumonisin levels (averaging 5490 mg/kg).     

Biosynthesis of lipid-linked oligosaccharides in embryonic liver. Formation of mannose containing derivatives

In the presence of exogenous dolichyl phosphate mannosyl transferase activity towards dolichyl phosphate was nearly 3-fold higher in microsomes from pig embryonic liver compared to that from adult liver. After incubation of microsomes from embryonic liver with UDP-N-acetylglucosamine and GDP-[14C]mannose lipid-linked tri- to undecasaccharides were discovered in CHCl3-CH3OH (2:1, v/v) and CHCl3-CH3OH-H2O (1:1:0.3, by vol) extracts. The main proportion of the radioactivity was incorporated into penta-, sexta and undecasaccharides. Amphomycin at concentration 500 micrograms/ml inhibited almost completely dolichyl phosphate mannose synthesis in embryonic liver microsomes without inhibition the formation of lipid-linked penta- and sextasaccharides. It was suggested that mannose transferred to lipid-linked tetra- to heptasaccharides comes from GDP-mannose but not from dolichyl phosphate mannose.     

Effects of Fusarium moniliforme and corn associated with equine leukoencephalomalacia on rat neurotransmitters and metabolites

Fusarium moniliforme (FM) is associated with equine leukoencephalomalacia (ELEM) and hepatotoxicities in horses and rats. The neurochemical effects of ELEM-associated corn naturally infected with FM and FM strain MRC 826 were studied in rats. Increases in brain 5-hydroxyindoleacetic acid (5-HIAA, major metabolite of serotonin, 5-HT) and 5-HIAA/5-HT ratios were observed in rats fed the ELEM-FM corn. These rats had reduced body weights (17%, P less than 0.01) and increased brain weight/body weight ratios (14%, P less than 0.01) as compared with controls that were fed commercial corn. Rats fed a rodent chow supplemented (16%, w/w) with corn cultures of FM (MRC 826) had brain 5-HT and 5-HIAA increased (11% and 60%, P less than 0.01, respectively). At 20% FM (MRC 826)-chow diet, the 5-HIAA levels were increased (18%, P less than 0.01). In both the 16% and 20% diets, brain 5-HIAA/5-HT ratios were increased (45%, P less than 0.01 and 10%, P less than 0.05), body weights reduced (30% and 18%, P less than 0.01) and brain weight/body weight ratios increased (40% and 16%, P less than 0.01), respectively. The incidences of microscopic liver lesions (particularly bile duct proliferations, hepatocellular hyperplasia, and focal necrosis) were consistent with rats fed the FM contaminated and FM-fortified diets. These results suggest a possible FM (ELEM-associated)-induced dysfunction in either 5-HT metabolism or 5-HIAA elimination in rat brains.     

Effects of Fusarium moniliforme culture extracts and fumonisin B1 on DNA,RNA and protein synthesis by baby hamster kidney cells

Baby hamster kidney cells (BHK-21) were exposed to culture filtrates of 4 Fusarium moniliforme isolates containing varying levels of fumonisin B1 (FMB1) and the effects upon RNA, DNA and protein synthesis were monitored. Cells were also grown on medium amended with FMB1 only for comparison. After 24 h incubation FMB1 (100 μg/100 ml medium) reduced protein synthesis by 4% and by 18% after 48 h. Culture filtrates containing the highest levels of FMB1 also caused the greatest inhibition in protein synthesis after 24 h but after 48 h protein synthesis levels were the same as controls even though the FMB1 level was 360 μg/100 ml. Only FMB1 reduced DNA synthesis, by 8% after 24 h but after 48 h DNA levels had increased by 40 % over controls. The culture filtrates containing the highest levels of FMB1 (360 μg/100 ml) reduced DNA synthesis more than 50% after 24 h and 48 h. Culture filtrates containing lesser amounts of FMB1 in some instances stimulated DNA synthesis and inhibited it in others. There was also no correlation in the level of FMB1 with the inhibition of RNA synthesis by BHK cells. It appears that metabolites other than fumonisin produced by F. moniliforme in culture can affect and both stimulate and inhibit RNA, DNA and protein synthesis by BHK cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of fumonisin B1 by Fusarium moniliforme NRRL 13616 in submerged culture

Fumonisin B1, a recently discovered mycotoxin, was synthesized by submerged cultures of Fusarium moniliforme NRRL 13616 grown for 29 days at 28 degrees C and 220 rpm in a basal salts medium (pH 5.0) supplemented with 90 g of glucose per liter and 3.5 g of ammonium sulfate per liter. Under these culture conditions, 74 +/- 23 micrograms of fumonisin B1 per ml was produced by 29-day-old F. moniliforme NRRL 13616 cultures. Fumonisin B1 was detected in liquid culture extracts by high-performance thin-layer chromatography. Fumonisin B1 was confirmed and quantitated by gas chromatography and gas chromatography-mass spectral analysis of the trimethylsilyl derivative. The use of a defined medium for producing fumonisin B1 in a submerged culture facilitates its isolation and provides an excellent method for conducting biosynthetic studies.     

Production of fumonisin B1 by Fusarium moniliforme NRRL 13616 in submerged culture.

Fumonisin B1, a recently discovered mycotoxin, was synthesized by submerged cultures of Fusarium moniliforme NRRL 13616 grown for 29 days at 28 degrees C and 220 rpm in a basal salts medium (pH 5.0) supplemented with 90 g of glucose per liter and 3.5 g of ammonium sulfate per liter. Under these culture conditions, 74 +/- 23 micrograms of fumonisin B1 per ml was produced by 29-day-old F. moniliforme NRRL 13616 cultures. Fumonisin B1 was detected in liquid culture extracts by high-performance thin-layer chromatography. Fumonisin B1 was confirmed and quantitated by gas chromatography and gas chromatography-mass spectral analysis of the trimethylsilyl derivative. The use of a defined medium for producing fumonisin B1 in a submerged culture facilitates its isolation and provides an excellent method for conducting biosynthetic studies.     

重楼属植物甾体皂甙的高效液相色谱分析

应用高效液相层析技术,对重楼属十八个种植物的甾体皂甙进行了定性、定量分析。植物用甲醇提取,抽出物经DIAION柱,以90％甲醇洗出总甙。在HPLC上,用ODS柱先将总甙以用醇:水(9:1)洗脱分为三馏段,每馏再在ODS柱或Rp-8柱上以甲醇:水(8:2;7:3.5)洗脱,使各个皂甙成分完全分离。被分离的每个色谱峰与已知重楼皂甙的保留时间进行比较并配合HPLC的加入法,TLC分析及用HPLC制备少量样品做MS测定来加以定性鉴定。定量采用内标及校正曲线法。     

Biosynthesis and characterization of lipid-linked sugars and glycoproteins in aorta.

A particulate enzyme from bovine aorta catalyzes the incorporation of mannose from GDP-D-[14C]mannose into three products as follows: 1. Most of the radioactivity which is incorporated in short term incubations is into a product that is soluble in CHCl3/CH3OH (2/1, v/v). This product was purified by chromatography on DEAE-cellulose and Sephadex LH-20. The purified glycolipid was stable to alkaline saponification but released [14C]mannose when subjected to mild acid hydrolysis (1/2 = 7 min at 100 degrees in 0.01 N HCl). The purified glycolipid had the same mobility on silica gel plates in an acidic, basic, or neutral solvent system as did glycolipid had the same mobility on silica gel plates in an acidic, basic, or neutral solvent system as did authentic dolichyl mannopyranosyl phosphate. The synthesis of the 14C-mannolipid was reversed by the addition of GDP and Mg2+. 2. [14C]mannose is also incorporated, although at a slower rate into products which are soluble in CHCl3/CH3OH/H2O (1/1/0.3, v/v). When the 1/10.3 soluble material was chromatographed on Avicel plates, it gave rise to three distinct radioactive bands which appear to be lipid-linked oligosaccharides. Mild acid hydrolysis of the 1/10.3 soluble material released water-soluble, neutral 14C-oligosaccharides which eluted from Sephadex G-50 in two or three peaks between the standards cytochrome c and GDP-mannose...     

甘肃黑蛋巢抗松梢枯病菌物质的分离纯化及抑菌活性*

采用MS培养基培养甘肃黑蛋巢Cyathus gansuensis,以生测为导向,乙酸乙酯和氯仿从甘肃黑蛋巢MS培养液中萃取出抗菌活性组分。Rp-18反相色谱柱和高效液相色谱仪相结合,从抗菌代谢产物分离纯化出一种抗松梢枯病菌(Sphaeropsis sapinea)的活性物质CXL-I,高效液相色谱检测表明CXL-I为一纯物质。CXL-I对松梢枯病菌的菌丝生长和孢子萌发有较强的抑制活性,当CXL-I浓度为50礸/ml时对菌丝生长抑制率可达80%,对孢子萌发抑制率达95%以上。