Dehydrocostuslactone and plant growth activity of derived guaianolides

The stereostructures of two new guaianolides, isodehydrocostuslactone and isozaluzanin C, isolated previously from Saussurea lappa, have been confirmed by their correlation with dehydrocostuslactone. Twenty new derivatives have been synthesized from these guaianolides and these have been tested as plant growth regulators. The conjugated lactones which have an exocyclic methylene group at C-4 conjugated with a C-3 ketone, show distinct enhancement in their root-forming potential, as compared with their 3-deoxy derivatives. Of further significance is the fact that these ketones display maximum activity only at lower concentrations. Other compounds show the expected structure-biological activity relationships displayed in general by guaianolides. However, the presence of an epoxide at the C-3, C-4 position does not affect the biological activity, which is indeed the case when the epoxide group occupies the C-4, C-14 position in guaianolides. The major biological parameter studied was rooting in-stem cuttings of Phaseolus aureus.     

Location of the epoxide function determines specificity of the allelic variants of human glutathione transferase Pi toward benzo[c]chrysene diol epoxide isomers

Carcinogenic activity of many polycyclic aromatic hydrocarbons (PAHs) is mainly attributed to their respective diol epoxides, which can be classified as either bay or fjord region depending upon the location of the epoxide function. The Pi class human glutathione (GSH) transferase (hGSTP1-1), which is polymorphic in humans with respect to amino acid residues in positions 104 (isoleucine or valine) and/or 113 (alanine or valine), plays an important role in the detoxification of PAH-diol epoxides. Here, we report that the location of the epoxide function determines specificity of allelic variants of hGSTP1-1 toward racemic anti-diol epoxide isomers of benzo[c]chrysene (B[c]C). The catalytic efficiency (k(cat)/K(m)) of V104,A113 (VA) and V104,V113 (VV) variants of hGSTP1-1 was approximately 2.3- and 1.7-fold higher, respectively, than that of the I104,A113 (IA) isoform toward bay region isomer (+/-)-anti-B[c]C-1,2-diol-3,4-epoxide. On the other hand, the IA variant was approximately 1.6- and 3.5-fold more efficient than VA and VV isoforms, respectively, in catalyzing the GSH conjugation of fjord region isomer (+/-)-anti-B[c]C-9,10-diol-11,12-epoxide. The results of the present study clearly indicate that the location of the epoxide function determines specificity of the allelic variants of hGSTP1-1 in the GSH conjugation of activated diol epoxide isomers of B[c]C.     

Mutagenicity of potentially carcinogenic mycotoxins produced byFusarium moniliforme

The mutagenic behaviour of two potentially carcinogenic mycotoxins produced byFusarium moniliforme was investigated in theSalmonella mutagenicity test using tester strains TA97a, TA98, TA100, and TA102. The mutagenic response obtained with fusarin C (1, 5, and 10μg/plate) against tester strains TA98 and TA100 in the presence of microsomal activation confirmed previous observations on the mutagenic behaviour of this mutagen while that obtained against TA97a is reported for the first time. No dose-response relationship could be detected for the concentration levels (0.2, 0.5, 1, 5, 10 mg/plate) tested for FB₁, FB₂, and FB₃ against any of the tester strains used in either the plate incorporation and / or the pre-incubation tests. A cytotoxic effect was obtained at concentration levels of 5 and 10mg/plate in the absence of the microsomal activation mixture. From the studies it became evident thatF moniliforme produces two compounds, a mutagenic compound, fusarin C which has been shown to lack carcinogenic activity in rats and the non-mutagenic fumonisin B mycotoxins of which FB₁ is known to be responsible for the hepatocarcinogenicity of the fungus in rats.     

Screening for Fusarin C production by European isolates of Fusarium species

A total of 137Fusarium isolates were screened forin vitro production of the mutagenic metabolite fusarin C, using a simple thin layer chromatographic method. It has been proven thatFusarium species (F. culmorum, F. graminearum, F. crookwellense, F. sporotrichioides, F. poae, F. tricinctum, andF. Avenaceum) isolated from European agricultural crops and soils are able to produce fusarin C. No fusarin C production was detected among isolates ofF. arthrosporioides, F. acuminatum, or F. equiseti. Results obtained by High-Performance Liquid Chromatography (HPLC) analyses of fungal extracts show that up to 26 chromatographic peaks having UV spectra similar to that of fusarin C are produced. It is not known if any of these metabolites are as mutagenic as fusarin C.     

Radiometric assays for mammalian epoxide hydrolases and glutathione S-transferase

A number of epoxides, including cis- and trans-stilbene oxides, were assayed as substrates for epoxide hydrolases (EHs) by gas-liquid chromatography. Radiolabeled stilbene oxides were prepared by sodium borotritide reduction of desyl chloride followed by ring closure with base treatment. Rapid radiometric assays for EHs were performed by differential partitioning of the epoxide into dodecane, while the product diol remained in the aqueous phase. Glutathione (GSH) transferase was similarly assayed by partitioning the epoxide and diol, if formed metabolically, into 1-hexanol, while the GSH conjugate was retained in the aqueous phase. The cytosolic EH rapidly hydrates the trans isomer while the cis is very poorly hydrated. In contrast, the cis is a better substrate for the microsomal EH than the trans. GSH transferase utilized both epoxides as substrates, but conjugation is faster with the cis isomer. Cytosolic EH activity is high in mouse but very low in rat and guinea pig. Microsomal EH activity, in contrast, is highest in guinea pig, intermediate in rat, and the lowest in mouse. GSH transferase activity, which is high in all three species, can be inhibited by chalcone, with an I₅₀ of 3.1 × 10^?5m. These assays facilitate the rapid evaluation and direct comparison of epoxide-metabolizing systems in cell homogenates used in short-term mutagenicity assays, cell or organ culture, and possibly in vivo.     

Carbasugar analogues of galactofuranosides: β-O-linked derivatives and towards β-S-linked derivatives

A selectively protected carbasugar analogue of β-galactofuranose was synthesised from glucose using ring-closing metathesis as the key step. The carbasugar was converted into an α-galacto configured 1,2-epoxide, which was an effective electrophile in Lewis acid catalysed coupling reactions with alcohols. The epoxide was opened with regioselective attack at C-1 to give β-galacto configured C-1 ethers. Using carbohydrates as nucleophiles, we synthesised a number of pseudodisaccharides. The epoxide was also regioselectively opened at C-1 with a sulfur nucleophile under basic conditions to give a β-galacto configured C-1 thioether.     

Identification and Regulation of fusA,the Polyketide Synthase Gene Responsible for Fusarin Production in Fusarium fujikuroi

Fusarins are a class of mycotoxins of the polyketide family produced by different Fusarium species, including the gibberellin-producing fungus Fusarium fujikuroi. Based on sequence comparisons between polyketide synthase (PKS) enzymes for fusarin production in other Fusarium strains, we have identified the F. fujikuroi orthologue, called fusA. The participation of fusA in fusarin biosynthesis was demonstrated by targeted mutagenesis. Fusarin production is transiently stimulated by nitrogen availability in this fungus, a regulation paralleled by the fusA mRNA levels in the cell. Illumination of the cultures results in a reduction of the fusarin content, an effect partially explained by a high sensitivity of these compounds to light. Mutants of the fusA gene exhibit no external phenotypic alterations, including morphology and conidiation, except for a lack of the characteristic yellow and/or orange pigmentation of fusarins. Moreover, the fusA mutants are less efficient than the wild type at degrading cellophane on agar cultures, a trait associated with pathogenesis functions in Fusarium oxysporum. The fusA mutants, however, are not affected in their capacities to grow on plant tissues.     

Screening survey of co-production of fusaric acid,fusarin C,and fumonisins B1, B2 and B3 by Fusarium strains grown in maize grains

Fusarium species isolated from Belgian maize were screened for their ability to produce fusarin C, fusaric acid, fumonisins B₁ (FB₁), FB₂ and FB₃ in maize grains. First, cultivation of Fusarium species in Myro liquid medium allowed overcoming the shortage of the standard of fusarin C on the market. All Fusarium verticillioides produced much higher contents of mycotoxins in Myro compared to Fusarium graminearum or Fusarium venenatum. The optimization of the LC-MS/MS method resulted in low limits of detection and quantification for fusarin C, fusaric acid, FB₁, FB₂ and FB₃ determination in maize grains. Its application for screening the potential toxin production ability evidenced that the concentrations of the analytes were significantly increased at various levels when F. verticillioides strains were cultivated in maize grains and reached 441 mg kg^?1 for fusaric acid, 74 mg kg^?1 for fusarin C, 1,301 mg kg^?1 for FB₁, 367 mg kg^?1 for FB₂ and 753 mg kg^?1 for FB₃.     

Preparative-scale kinetic resolution of racemic styrene oxide by immobilized epoxide hydrolase

Epoxide hydrolase from Aspergillus niger was immobilized onto the modified Eupergit C 250 L through a Schiff base formation. Eupergit C 250 L was treated with ethylenediamine to introduce primary amine groups which were subsequently activated with glutaraldehyde. The amount of introduced primary amine groups was 220 μmol/g of the support after ethylenediamine treatment, and 90% of these groups were activated with glutaraldehyde. Maximum immobilization of 80% was obtained with modified Eupergit C 250 L under the optimized conditions. The optimum pH was 7.0 for the free epoxide hydrolase and 6.5 for the immobilized epoxide hydrolase. The optimum temperature for both free and immobilized epoxide hydrolase was 40 °C. The free epoxide hydrolase retained 52 and 33% of its maximum activity at 40 and 60 °C, respectively after 24 h preincubation time whereas the retained activities of immobilized epoxide hydrolase at the same conditions were 90 and 75%, respectively. Immobilized epoxide hydrolase showed about 2.5-fold higher enantioselectivity than that of free epoxide hydrolase. A preparative-scale (120 g/L) kinetic resolution of racemic styrene oxide using immobilized preparation was performed in a batch reactor and (S)-styrene oxide and (R)-1-phenyl-1,2-ethanediol were both obtained with about 50% yield and 99% enantiomeric excess. The immobilized epoxide hydrolase was retained 90% of its initial activity after 5 reuses.     

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

Glutathione (GSH) exerted a profound effect on the oxidation of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and 7-benzyloxyquinoline (BQ) by human liver microsomes as well as by CYP3A4-containing insect cell microsomes (Baculosomes). The cooperativity in O-debenzylation of both substrates is eliminated in the presence of 1-4 mM GSH. Addition of GSH also increased the amplitude of the 1-PB induced spin shift with purified CYP3A4 and abolished the cooperativity of 1-PB or BFC binding. Changes in fluorescence of 6-bromoacetyl-2-dimethylaminonaphthalene attached to the cysteine-depleted mutant CYP3A4(C58,C64) suggest a GSH-induced conformational changes in proximity of α-helix A. Importantly, the K_S value for formation of the GSH complex and the concentrations in which GSH decreases CYP3A4 cooperativity are consistent with the physiological concentrations of GSH in hepatocytes. Therefore, the allosteric effect of GSH on CYP3A4 may play an important role in regulation of microsomal monooxygenase activity in vivo.     

Modification of glutathione levels in C3H/10T1/2 cells and its relationship to benzo(a)pyrene anti-7,8-dihydrodiol 9,10-epoxide-induced cytotoxicity

Levels of reduced glutathione (GSH) in C3H/10T1/2 cells were selectively altered to determine what quantitative role GSH transferase-catalyzed conjugation plays in regulating the cytotoxic effects of benzo(a)pyrene anti-7,8-dihydrodiol 9,10-epoxide (r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene, anti-diol epoxide). A 65% decrease in 10T1/2 cell GSH content from 0.16 mM (control cell GSH concentration) to 0.06 mM was accompanied by a 46% decrease in the anti-diol epoxide LD80; a 98% increase in GSH content resulted in a 44% increase in anti-diol epoxide LD80. This nonlinear relationship between changes in cellular GSH concentration and anti-diol epoxide LD80 was directly relatable to the nonlinear change in the rate of anti-diol epoxide conjugation which was catalyzed by 10T1/2 cell GSH transferases. Purified 10T1/2 cell cytosol catalyzed the GSH conjugation of anti-diol epoxide to yield a GSH conjugation product with a distinct UV absorbance spectrum; the apparent GSH Km for this cell cytosol-catalyzed reaction was 0.20 mM. Variations in the cellular GSH concentration around the GSH Km resulted in a nonlinear change in the amount of anti-diol epoxide-GSH conjugate formed, and a reciprocal change in the amount of free anti-diol epoxide available for cytotoxic alkylation events. These results clarify in quantitative, biochemical terms how GSH transferase-catalyzed conjugation can regulate the level of an electrophilic carcinogen metabolite in a biological system.     

Localization of epoxide-metablozing enzymes in rat testis

Antibodies raised against rat hepatic epoxide hydrolase (EC 3.3.2.3) and glutathione S-transferases (EC 2.5.1.18) B, C and E were used to determine the presence and localizations of these epoxide-metabolizing enzymes in testes of sexually immature and mature Wistar and Holtzman rats. Unlabeled antibody peroxidase-antiperoxidase staining for each enzyme was readily detected in rat testes at the light microscopic level. Although significant strain-related differences were not apparent, staining intensity for certain enzymes differed markedly between Leydig cells and seminiferous tubules. Leydig cells of immature and mature rats were stained much intensely for epoxide hydrolase and glutathione S-transferase B and E than were seminiferous tubules, whereas Sertoli cells, spermatogonia, spermatocytes and spermatids, as well as Leydig cells, were stained intensely by the anti-glutathione S-transferase C. Age-related differences in staining for glutathione S-transferase B were not obvious, while the anti-glutathione S-transferase C stained seminiferous tubules more intensely in immature rats, and antibodies to expoxide hydrolase and glutathione S-transferases C and E stained Leydig cells much more intensely in mature rats. These observations thus demonstrate that testes of both sexually immature and mature rats contain epoxide hydrolase and glutathione S-transferases. Except for glutathione S-transferase C in immature rats, Leydig cells appear to contain much higher levels of enzymes than do seminiferous tubules. During sexual maturation, the testicular level of glutathione S-transferase B appears to remain constant, while levels of epoxide hydrolase and glutathione S-transferases C and E increase within Leydig cells and the level of glutathione S-transferase C decreases within seminiferous tubules.     

Biosynthesis of salicylic acid in fungus elicited Catharanthus roseus cells

Feeding experiments using [1-¹³C]-d-glucose to Catharanthus roseus (L.) G.Don cell suspension cultures followed by elicitation with Pythium aphanidermatum extract were performed in order to study the salicylic acid (SA) biosynthetic pathway and that of 2,3-dihydroxybenzoic acid (2,3-DHBA) as a comparison. A strongly labeled C-7 and a symmetrical partitioning of the label between C-2 and C-6 would occur if SA was synthesized from phenylalanine. In case of the isochorismate pathway, a relatively lower incorporation at C-7 and a non-symmetrical incorporation at C-2 and C-6 would be obtained. Relatively, high- and non-symmetrical enrichment ratios at C-2 and C-6, and a lower enrichment ratio at C-7 were observed in both SA and 2,3-DHBA detected by ¹³C NMR inverse gated spectrometry leading to the conclusion that the isochorismate pathway is responsible for the biosynthesis of both compounds. However, different enrichment ratios of the labeled carbons in SA and 2,3-DHBA indicate the use of different isochorismate pools, which means that their biosynthesis is separated in time and/or space.     

Activité comparée des hormones juvéniles en C-18 (JH-I) et en C-16 (JH-III) chez Locusta migratoria

The comparative activity of C-16 and C-18 juvenile hormones is studied in Locusta migratoria on four well-known physiological functions of the corpora allata by means of a single injection of a solution of hormone in oil at doses of 50, 100, and 200 μg/animal. Judged on morphogenesis and pigmentation, JH-I (C-18 JH) as well as JH-III (C-16 JH) show a real juvenilizing effect. The potency of JH-I is much higher than that of JH-III because the first hormone only produces supernumerary larvae and most modified green animals. JH-I counterbalances exactly the lack of CA on the gonadotropic function whereas JH-III allows only about 50 per cent development of oöcytes. The cardiotropic activity of JH-I is similar to that of the CA. The C-18 juvenile hormone is until now the only studied ‘juvenilizing’ compound which increases the heartbeat. JH-III appears to have no noticeable effect on the heart.These results combine to prove that only JH-I has an activity similar to the Locusta corpora allata on morphogenesis, pigmentation, ovarian maturation, and the cardiac activity of L. migratoria.     

Production of fusarin C on cereal and soybean by Fusarium moniliforme

Two isolates of Fusarium moniliforme were compared with respect to production of a mutagenic compound, fusarin C, on seven corn varieties as well as on soybean, wheat, rye, barley, and a liquid culture medium. The isolates were originally obtained from corn and barley. Both isolates produced fusarin C on seed of all five crops within a 21-day period, and one isolate produced the largest amount on oats. Soybean was the poorest substrate for both isolates. Although the quantity of fusarin C produced on grain was isolate dependent, specific substrate requirements for each strain were suggested. The isolates differed in their ability to grow and produce fusarin C on corn with different moisture contents (16, 20, 24, and 28%). One isolate was more xerotolerant and grew at 16% moisture but did not produce the mutagen.     

Metabolic activation and hepatotoxicity. Toxicity of bromobenzene in hepatocytes isolated from phenobarbital-and diethylmaleate-treated rats.

Hepatocytes freshly isolated from diethylmaleate-treated rats exhibited a markedly decreased concentration of reduced glutathione (GSH) which increased to the level present in hepatocytes from nontreated rats upon incubation in a complete medium. When bromobenzene was present in the medium, however, this increase in GSH concentration upon incubation was reversed and a further decrease occurred that resulted in GSH depletion and cell death. This was prevented by metyrapone, an inhibitor of the cytochrome P-450-linked metabolism of bromobenzene. Bromobenzene metabolism in hepatocytes was accompanied by a fraction of metabolites covalently binding to cellular proteins. The size of this fraction, relative to the amount of total metabolites, was increased in hepatocytes isolated from diethylmaleate-treated rats and in hepatocytes from phenobarbital-treated rats incubated with bromobenzene in the presence of 1,2-epoxy-3,3,3-trichloropropane, an inhibitor of microsomal epoxide hydrase which, however, also acted as a GSH-depleting agent. In addition, the metabolism of bromobenzene by hepatocytes was associated with a marked decrease in various coenzyme levels, including coenzyme A, NAD(H), and NADP(H). Cysteine and cysteamine inhibited the formation of protein-bound metabolites of bromobenzene in microsomes, but did not prevent bromobenzene toxicity in hepatocytes when added at higher concentrations to the incubation medium (containing 0.4 mm cysteine). Methionine, on the other hand, did not cause a significant effect on bromobenzene metabolism in microsomes and prevented toxicity in hepatocytes, presumably by stimulating GSH synthesis and thereby decreasing the amount of reactive metabolites available for interaction with other cellular nucleophiles. It is concluded that, in contrast to hepatocytes with normal levels of GSH, hepatocytes from diethylmaleate-treated rats were sensitive to bromobenzene toxicity under our incubation conditions. In this system, bromobenzene metabolism led to GSH depletion and was associated with a progressive decrease in coenzyme A and nicotinamide nucleotide levels and a moderate increase in the formation of metabolites covalently bound to protein. Methionine was a potent protective agent which probably acted by enhanced GSH synthesis via the formation of cystathionine.     

On the mechanism of conversion of dethiobiotin to biotin in Escherichia coli. Discussion of the occurrence of an intermediate hydroxylation.

The last step of the biosynthesis of biotin, i.e. the conversion of dethiobiotin to biotin was studied using E. coli. The three dethiobiotin derivatives hydroxylated at C-2 or C-5 were synthesized and tested as potential precursors of biotin. It appears that none of these compounds is able to support the growth of E. coli C124, a mutant which does not synthesize dethiobiotin, but converts it into biotin. These results strongly disfavour the hypothesis of the activation of the saturated carbons by an hydroxylation process.     

A new mycinosyl rosamicin derivative produced by an engineered Micromonospora rosaria mutant with a cytochrome P450 gene disruption introducing the d-mycinose biosynthetic gene

Genetic engineering of post-polyketide synthase-tailoring genes can be used to generate new macrolide analogs through manipulation of the genes involved in their biosynthesis. Rosamicin, a 16-member macrolide antibiotic produced by Micromonospora rosaria IFO13697, contains a formyl group and an epoxide at C-20 and C-12/13 positions which are formed by the cytochrome P450 enzymes RosC and RosD, respectively. The d-mycinose biosynthesis genes in mycinamicin II biosynthesis gene cluster of Micomonospora guriseorubida A11725 were introduced into the rosC and rosD disruption mutants of M. rosaria IFO13697. The resulting engineered strains, M. rosaria TPMA0054 and TPMA0069, produced mycinosyl rosamicin derivatives, IZIV and IZV, respectively. IZIV was identified as a novel mycinosyl rosamicin derivative, 23-O-mycinosyl-20-deoxo-20-dihydrorosamicin.     

Production of fusarin C on cereal and soybean by Fusarium moniliforme.

Two isolates of Fusarium moniliforme were compared with respect to production of a mutagenic compound, fusarin C, on seven corn varieties as well as on soybean, wheat, rye, barley, and a liquid culture medium. The isolates were originally obtained from corn and barley. Both isolates produced fusarin C on seed of all five crops within a 21-day period, and one isolate produced the largest amount on oats. Soybean was the poorest substrate for both isolates. Although the quantity of fusarin C produced on grain was isolate dependent, specific substrate requirements for each strain were suggested. The isolates differed in their ability to grow and produce fusarin C on corn with different moisture contents (16, 20, 24, and 28%). One isolate was more xerotolerant and grew at 16% moisture but did not produce the mutagen.     

Fusarin C production by North American isolates of Fusarium moniliforme

A liquid culture medium was developed to screen North American isolates of Fusarium moniliforme Sheldon and Fusarium subglutinans (Wollenw. and Reink.) Nelson, Toussoun, and Marasas for their ability to produce fusarin C. Parameters which were important for the optimal biosynthesis of fusarin C included pH (3.0 to 4.0), aeration, and sugar concentration (30 to 40%). Of seven sugars tested, sucrose and glucose were the best carbohydrate sources for mycotoxin production, resulting in levels of fusarin C of greater than 60 ppm (greater than 60 micrograms/g) in liquid culture (28 degrees C; 7 days). A time-course study of fusarin C production was done over a 21-day period, during which time pH values, glucose concentrations, nitrogen levels, and fungal biomass were determined. Of the two Fusarium spp. studied, 13 of 16 isolates of F. moniliforme produced fusarin C in liquid medium (14 of 16 in corn), while none of the 15 isolates of F. subglutinans studied was found to produce the compound. Levels of fusarin C produced by Fusarium sp. isolates growing on corn ranged from 18.7 to 332.0 micrograms/g.