Steroid transformations with Fusarium oxysporum var. cubense and Colletotrichum musae

The utility of two locally isolated fungi, pathogenic to banana, for steroid biotransformation has been studied. The deuteromycetes Fusarium oxysporum var. cubense (IMI 326069, UAMH 9013) and Colletotrichum musae (IMI 374528, UAMH 8929) had not been examined previously for this potential. In general, F. oxysporum var. cubense effected 7alpha hydroxylation on 3beta-hydroxy-delta5-steroids, 6beta, 12beta, and 15alpha hydroxylation on steroidal-4-ene-3-ones, side-chain degradation on 17alpha,21-dihydroxypregnene-3,20-diones, and 15alpha hydroxylation on estrone. Both strains were shown to perform redox reactions on alcohols and ketones.     

Optimization of extracellular fungal peroxidase production by 2 Coprinus species

Optimum culture conditions for the batch production of extracellular peroxidase by Coprinus cinereus UAMH 4103 and Coprinus sp. UAMH 10067 were explored using 2 statistical experimental designs, including 2-level, 7-factor fractional factorial design and 2-factor central composite design. Of the 7 factors examined in the screening study, the concentrations of carbon (glucose) and nitrogen (peptone or casitone) sources showed significant effects on the peroxidase production by Coprinus sp. UAMH 10067. The optimum glucose and peptone concentrations were determined as 2.7% and 0.8% for Coprinus sp. UAMH 10067, and 2.9% and 1.4% for C. cinereus UAMH 4103, respectively. Under the optimized culture condition the maximum peroxidase activity achieved in this study was 34.5 U x mL(-1) for Coprinus sp. UAMH 10067 and 68.0 U x mL(-1) for C. cinereus UAMH 4103, more than 2-fold higher than the results of previous studies.     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.     

Purification, characterization and evaluation of extracellular peroxidase from two Coprinus species for aqueous phenol treatment

Non-ligninolytic fungal peroxidases produced by Coprinus cinereus UAMH 4103 and Coprinus sp. UAMH 10067 were purified, characterized and evaluated as cost-effective alternatives to horseradish peroxidase for aqueous phenol treatment. Purified Coprinus peroxidases exhibited a molecular weight of 36 kDa on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Although the catalytic properties of the two Coprinus peroxidases were nearly identical in both crude and purified forms, the stabilities were substantially different. The peroxidase from Coprinus sp. UAMH 10067 was more stable at 50 degrees C and under basic conditions (up to pH 10) than the enzyme from C. cinereus UAMH 4103. The former enzyme also performed better at pH 9 than the latter one in aqueous phenol treatment. The phenol removal efficiency of the Coprinus peroxidase was comparable to those of previously studied plant peroxidases. The broader working pH and higher thermal and alkaline stability of the peroxidase from Coprinus sp. UAMH 10067 may be advantageous for its application to industrial wastewater treatment.     

Extracellular peroxidase production by Coprinus species from urea-treated soil

Thirteen strains of inky-cap mushroom Coprinus species were evaluated for the production of extracellular peroxidase. The liquid fermentation was carried out in shake flasks containing 1% glucose, 0.5% peptone, 0.3% yeast extract, and 0.3% malt extract broth at 25 degrees C. Peroxidase activity was detected in the liquid culture of several Coprinus species, including C. echinosporus NBRC 30630; C. macrocephalus NBRC 30117; Coprinus spp. UAMH 10065, UAMH 10066, UAMH 10067, and 074, after 10 days of growth. Peroxidase production by Coprinus sp. UAMH 10067, a Coprinus species isolated from urea-treated soil, was comparable to that of C. cinereus and reached 15 U.mL(-1) after 10 days. In addition, the peroxidase from Coprinus sp. UAMH 10067 was apparently more thermally stable than the enzyme produced by C. cinereus.     

Effect of growth conditions on the production of manganese peroxidase by three strains of Bjerkandera adusta

We were looking for a strain of Bjerkandera adusta that produces high titres of manganese peroxidase under optimal conditions for large-scale enzyme purification. We have chosen two strains from the University of Alberta Microfungus Collection and Herbarium, UAMH 7308 and 8258, and compared the effects of growth conditions and medium composition on enzyme production with the well-characterized strain BOS55 (ATCC 90940). Of four types of cereal bran examined, rice bran at 3% (w/v) in 60 mM phosphate buffer pH 6 supported the highest levels of enzyme production. Using 100 mL medium in 500-mL Erlenmeyer flasks, maximum enzyme levels in the culture supernatant occurred after about 10 days of growth; 5.5 U x mL(-1) for UAMH 7308, 4.4 U x mL(-1) for UAMH 8258, and 1.7 U x mL(-1) for BOS55, where units are expressed as micromoles of Mn-malonate formed per minute. Growth as submerged cultures in 10-L stirred tank reactors produced 3.5 U x mL(-1) of manganese peroxidase (MnP) by UAMH 8258 and 2.5 U x mL(-1) of MnP by 7308, while enzyme production by BOS55 was not successful in stirred tank reactors but could be scaled up in 2-L shake flasks containing 400 mL rice bran or glucose-malt-yeast extract (GMY)-Mn-glycolate medium to produce MnP levels of 1.7 U x mL(-1). These results show that the two strains of B. adusta, UAMH 7308 and 8258, can produce between two and three times the manganese peroxidase level of B. adusta BOS55, that they are good candidates for scale up of enzyme production, and that the rice bran medium supports higher levels of enzyme production than most previously described media.     

Deoxyribonucleic acid hybridization studies of Exophiala dermatitidis and Exophiala jeanselmei

Exophiala dermatitidis and Exophiala jeanselmei share similar morphological features and have been confused with each other. To clarify the relationship between the two fungi, we conducted a deoxyribonucleic acid (DNA)-DNA hybridization study using a dot blot method. Between E. dermatitidis and E. jeanselmei, only a very low level of DNA relatedness was seen and it was confirmed that these two fungi are distinct species based on DNA similarity. Close correspondence of DNA from the isolates of E. dermatitidis was obtained, whereas the isolates of E. jeanselmei were divided into 6 groups according to their DNA similarity and a possibility was shown that E. jeanselmei is composed of genetically heterogeneous groups. The subdivision of the species E. jeanselmei by the DNA-DNA hybridization method was in agreement with serotyping exoantigens. This result suggests that DNA-DNA hybridization studies provide an excellent tool for the identification and grouping of pathogenic dematiaceous fungi.     

棘状外瓶霉核糖体基因的初步研究

外瓶霉可致人类感染 ,不同生物群落的菌种 ,其致病性、药敏性等特征具有差异性。通过对 1 0株棘状外瓶霉核糖体基因及其转录间隔区进行序列测定 ,并与GeneBank中 9株同种真菌对比分析 ,揭示了不同生物群落的棘状外瓶霉虽然形态学差异性小 ,但在基因学上具有差异性 ;原属于甄氏外瓶霉变种的BMU 0 0 0 45 7(ATCC 2 41 5 2E .jeanselmeivar.hetoromorpha)与 2株棘状外瓶霉具有 1 0 0 %的同源性。研究提示了形态学特征相似的棘状外瓶霉在基因水平上具有差异性 ,核糖体基因及其转录间隔区对于研究菌群特性具有一定意义。     

Degradation of ethanol plant by-products by <Emphasis Type="Italic">Exophiala lecanii-corni</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in batch studies

By-product emissions from ethanol production facilities have become a public health concern. Many of these by-products are classified as hazardous air pollutants by the USEPA and current treatment methods, mainly thermal-oxidation, for these compounds are costly, energy intensive, and may produce other undesirable by-products. Degradation of these by-products by the fungi Exophiala lecanii-corni and Saccharomyces cerevisiae was explored. Ethanol plant by-products, focused on in this study, included formaldehyde, acetaldehyde, ethanol, methanol, glycerol, acetic acid, and lactic acid. Initial batch studies were conducted to determine degradation rates and whether the contaminants would be toxic to the fungi. These batch studies demonstrated that E. lecanii-corni and S. cerevisiae are able to utilize all but methanol and formaldehyde as sole carbon and energy sources for growth; however, both contaminants were utilized as secondary metabolites by cultures initially fed either ethanol or acetic acid. Growth studies also were conducted using two contaminants simultaneously to determine if the presence of one contaminant inhibited the degradation of another. Growth and contaminant utilization was observed in cultures fed two contaminants simultaneously.     

Biochemical and ultrastructural changes in the ovaries of red palm weevil,Rhynchophorus ferrugineus (Coleoptera: Curculionidae) following acute imidacloprid poisoning

This study aims to inspect the effects of Imidacloprid (IMI) acute toxicity in the ovaries of the red palm weevil (Rhynchophorus ferrugineus). The LC₅₀ of IMI was determined to be 15.7 ppm. We assessed the biochemical changes resulting from treatment with four IMI concentrations (10, 15, 20 and 30 ppm). IMI reduced acetylcholine esterase activity in a dose-dependent manner. Glutathione (GSH) content decreased with increasing concentration of IMI; however, GSH was not affected and accompanied by an increase in catalase activity with the highest concentration of IMI. Lipid peroxidation increased steadily as IMI concentration increased. Based on the ultrastructural analyses of the follicle cells and the ooplasm of developing oocytes, acute IMI toxicity produced damaged yolk bodies, pale nuclei, and shrinking microvilli. Fractionation of rough endoplasmic reticulum, deformed mitochondria, and autophagic bodies were also observed. In conclusion, these ovarian pathobiochemical changes could be used as biomarkers of acute IMI exposure.     

Susceptibility of the Hymenopteran Parasitoids Apoanagyrus ( = Epidinocarsis ) lopezi (Encyrtidae) and Phanerotoma sp. (Braconidae) to the Entomopathogenic Fungus Metarhizium anisopliae var. acridum (Deuteromycotina: Hyphomycetes)

Laboratory and so-called extended laboratory bioassays were conducted in Benin, West Africa, to investigate the pathogenicity and virulence of the entomopathogenic fungus Metarhizium anisopliae var. acridum , a biocontrol agent against locusts and grasshoppers, to two hymenopteran parasitoids, Apoanagyrus ( = Epidinocarsis ) lopezi and Phanerotoma sp. Treatments were carried out under simulated field conditions at standard field dose rates of 2.5 and 5.0 ×10 12 conidia ha -1 . Test organisms were continuously (3 weeks) exposed to spray residues in artificial or simulated natural environments. The standard strain IMI 330 189 of the mycopesticide Green Muscle caused a significant reduction of 24% in the longevity ( = average survival time, AST) of A. lopezi , relative to the untreated control. Mycosis was confirmed in 16% of all cadavers. AST was shorter under low relative humidity (RH) conditions, and these conditions seemed to enhance susceptibility to fungal infection. However, this effect was only marginally significant. In contrast, average longevity of untreated A. lopezi was slightly yet significantly shorter at low RH (50-60%) than at high RH (80-90%). In the extended laboratory assay, the same isolate had no significant effect on mortality, parasitoid emergence ( = beneficial capacity) and sex ratio. In a further screening test with isolates IIBC I91 609, IIBC I93 833 and IMI 330 189 (reference), no infection of A. lopezi was confirmed. Similarly, Phanerotoma sp. was not susceptible to IMI 330 189. It is concluded that mycopesticides based on the three strains tested pose a low risk to parasitic hymenopterans under field conditions.     

Allosteric Interaction Between the Site Labeled by [3H]Imipramine and the Serotonin Transporter in Human Platelets

The nature of interaction between the site labeled by [3H]imipramine (IMI) and the 5-hydroxytryptamine (5-HT, serotonin) transporter in human platelets was examined. The sulfhydryl characterizing agent N-ethylmaleimide (NEM) differentially affected [3H]5-HT uptake and [3H]IMI binding in human platelet preparations. Concentrations of NEM that completely abolished [3H]5-HT uptake only minimally reduced [3H]IMI binding. Examining the effect of IMI on the kinetics of human platelet [3H]5-HT uptake revealed significant reductions in maximal velocity (Vmax) without altering affinity (Km). IC50 values for selected uptake blockers on [3H]IMI binding and [3H]5-HT uptake were determined. IC50 values of these compounds for uptake and binding revealed that agents such as IMI, chlorpromazine, amitriptyline, and nisoxetine were preferential inhibitors of [3H]IMI binding whereas fluoxetine, CL 216, 303, pyrilamine, and bicifadine were preferential [3H]5-HT uptake blockers. 5-HT was a weak displacer of [3H]IMI binding (IC25 = 3.0 microM) and exhibited a rather low Hill coefficient (nH app = 0.46). Results reported herein support the notion of an allosteric interaction between the [3H]IMI binding site and the 5-HT transporter complex in human platelets.     

Studies on the phylogenesis of pathogenic ‘black yeasts’

This paper deals mainly with the conidium ontogenesis and phylogenesis of black yeasts such as E. jeanselmei, E. gougerotii, E. dermatitidis and E. spinifera. The conidium ontogenesis of E. jeanselmei, E. gougerotii and E. dermatitidis was almost the same. One to five annellated tips were observed through a scanning microscope at the apices of conidiogenous cells, which were bottle- or jar-shaped. Annellations on the tips looked like fringes and the conidiogenous cells of these three species were annellides. Annellated projections occurred on hyphae and annelloconidia were also produced from them. Occasionally, secondary annellides occurred from primary ones. They looked like moniliform hyphae. Daughter conidia sometimes budded directly from mother cells. The shapes and sizes of the conidia of these species were very similar to each other. The conidium ontogenesis of E. spinifera was annellidic as well. However, a single annellated tip usually occurred on an annellide. The annellated tips of the fungus were long and more than 20 annellations were observed on their walls. The conidiogenesis of the four species of Exophiala is only annellidic. There were no differences in the biological examinations except KNO3 assimilation among these four species. The growth of E. jeanselmei and E. gougerotii was poor at 37 degrees C. The GC contents of E. jeanselmei 1171, E. gougerotii B-1800, E. dermatitidis MM-7 and E. spinifera DU-3342 were 54.6, 54.6, 56.6 and 59.7%, respectively.     

Microbial hydroxylation of imidacloprid for the synthesis of highly insecticidal olefin imidacloprid

Microorganisms that bring about the aerobic transformation of imidacloprid (IMI) were isolated and screened, and the microbial regio- and stereoselective hydroxylation of IMI was studied. Some bacteria and fungi transformed IMI to 5-hydroxyl IMI. Bacterium Stenotrophomonas maltophilia CGMCC 1.1788 resting cells transformed IMI into R-5-hydroxyl IMI at the highest conversion rate. The enzyme catalyzed the stereoselective hydroxylation at position C12 of IMI in the imidazolidine ring. Under acidic conditions, 5-hydroxyl IMI was converted into olefin IMI in high molar conversion yield. The olefin IMI exhibited about 19 and 2.2 times more insecticidal efficacy than IMI against horsebean aphid imago and nymph, respectively, and about 1.4 times more active than IMI against brown planthopper imago. The transformation rate of IMI by resting cells of S. maltophilia CGMCC 1.1788 was promoted significantly by some carbohydrates and organic acids. The reaction medium with 5% sucrose resulted in 8.3 times greater biotransformation yield as compared with that without sucrose.     

Chronopharmacokinetics of Imipramine and Desipramine in Rat Forebrain and Plasma After Single and Chronic Treatment with Imipramine

Daily variations in the pharmacokinetics of imipramine (IMI) could contribute to circadian phase-dependent effects of the drug. Therefore, the chronopharmacokinetics of IMI and its metabolite, desipramine (DMI), were studied after single and chronic application. Male rats were synchronized to a 12:12 hour lightdark (L:D) regimen with lights on from 07:00 to 19:00 (dark, 19:00-07:00). In single-dose experiments rats were injected with IMI (10 mg/kg) i.p. or i.v. at 07:30 or 19:30 and groups of rats were killed 0-22 hours thereafter. After chronic application of IMI in drinking water (≈ 15 mg/kg/d) groups of rats were killed during the 14th day of treatment at 02:00, 08:00, 14:00, and 20:00, respectively. Brain and plasma concentrations of IMI and DMI were determined by reversed-phase high-performance liquid chromatography with ultraviolet detection. After single i.p. application of IMI, maximal brain concentrations (C_max) of IMI and DMI were nearly twofold higher in darkness (IMI, 4.8 μg/g; DMI, 1.8 μg/g) than in light (IMI, 2.85 Mg/g; DMI, 0.85 Mg/g). Also, the area under the curve (AUC) (0-22 hours) was about 1.6-fold greater in darkness than in light for IMI and DMI; half-lives were not circadian phase dependent. After i.v. injection of IMI, the AUC in brain was also about 30% greater in darkness than in light. After chronic application of IMI in drinking water, brain concentrations of IMI and DMI varied more than threefold within 24 hours. The data demonstrate that the pharmacokinetics of IMI and DMI are circadian phase dependent. It is assumed that circadian variations in drug distribution are more likely to contribute to the drug's chronopharmacokinetics than variations in the drug's metabolism. The 24-hour variations in the drug's concentrations after chronic IMI application in drinking water can be explained by the drinking behavior of the rats, which by itself is altered by IMI.     

Dibenzyl sulfide metabolism by white rot fungi

Microbial metabolism of organosulfur compounds is of interest in the petroleum industry for in-field viscosity reduction and desulfurization. Here, dibenzyl sulfide (DBS) metabolism in white rot fungi was studied. Trametes trogii UAMH 8156, Trametes hirsuta UAMH 8165, Phanerochaete chrysosporium ATCC 24725, Trametes versicolor IFO 30340 (formerly Coriolus sp.), and Tyromyces palustris IFO 30339 all oxidized DBS to dibenzyl sulfoxide prior to oxidation to dibenzyl sulfone. The cytochrome P-450 inhibitor 1-aminobenzotriazole eliminated dibenzyl sulfoxide oxidation. Laccase activity (0.15 U/ml) was detected in the Trametes cultures, and concentrated culture supernatant and pure laccase catalyzed DBS oxidation to dibenzyl sulfoxide more efficiently in the presence of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) than in its absence. These data suggest that the first oxidation step is catalyzed by extracellular enzymes but that subsequent metabolism is cytochrome P-450 mediated.     

Chronopharmacokinetics of imipramine and desipramine in rat forebrain and plasma after single and chronic treatment with imipramine.

Daily variations in the pharmacokinetics of imipramine (IMI) could contribute to circadian phase-dependent effects of the drug. Therefore, the chronopharmacokinetics of IMI and its metabolite, desipramine (DMI), were studied after single and chronic application. Male rats were synchronized to a 12:12 hour light:dark (L:D) regimen with lights on from 07:00 to 19:00 (dark, 19:00-07:00). In single-dose experiments rats were injected with IMI (10 mg/kg) i.p. or i.v. at 07:30 or 19:30 and groups of rats were killed 0-22 hours thereafter. After chronic application of IMI in drinking water (approximately 15 mg/kg/d) groups of rats were killed during the 14th day of treatment at 02:00, 08:00, 14:00, and 20:00, respectively. Brain and plasma concentrations of IMI and DMI were determined by reversed-phase high-performance liquid chromatography with ultraviolet detection. After single i.p. application of IMI, maximal brain concentrations (Cmax) of IMI and DMI were nearly twofold higher in darkness (IMI, 4.8 micrograms/g; DMI, 1.8 micrograms/g) than in light (IMI, 2.85 micrograms/g; DMI, 0.85 microgram/g). Also, the area under the curve (AUC) (0-22 hours) was about 1.6-fold greater in darkness than in light for IMI and DMI; half-lives were not circadian phase dependent. After i.v. injection of IMI, the AUC in brain was also about 30% greater in darkness than in light. After chronic application of IMI in drinking water, brain concentrations of IMI and DMI varied more than threefold within 24 hours. The data demonstrate that the pharmacokinetics of IMI and DMI are circadian phase dependent. It is assumed that circadian variations in drug distribution are more likely to contribute to the drug's chronopharmacokinetics than variations in the drug's metabolism. The 24-hour variations in the drug's concentrations after chronic IMI application in drinking water can be explained by the drinking behavior of the rats, which by itself is altered by IMI.     

Enhanced hydroxylation of imidacloprid by <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> upon addition of sucrose

Sucrose’s ability to promote the hydroxylation of imidacloprid (IMI) by bacterium Stenotrophomonas maltophilia strain CGMCC 1.1788 was examined. Both growing culture and resting cells could transform IMI into 5-hydroxy IMI. Adding 2% sucrose to the growing culture transformation broth and 5% sucrose to the resting cell transformation broth resulted in biotransformation yields, respectively, 2.5 and 9 times greater than without sucrose. In the growing culture transformation, sucrose increased biomass, which led to enhance hydroxylation of IMI. In the resting cell transformation, sucrose was used not as a carbon source but as an energy source for cofactor regeneration for hydroxylation of IMI. The hydroxylation activity of IMI was promoted eightfold by adding reduced nicotinamide adenine dinucleotide (NADH) to the cell-free extract. The hydroxylation of IMI was significantly inhibited by P450 inhibitor piperonyl butoxide. It seems that the hydroxylation of IMI by S. maltophilia CGMCC 1.1788 might proceed through a system by cooperating with P450 enzyme.     

Effects of imipramine on tyrosine and tryptophan are mediated by beta-adrenoceptor stimulation

Imipramine (IMI; 20 mg/kg) in rats decreased the plasma tyrosine concentration by 21% (90 min), whereas norepinephrine (NE; 1.25 mg/kg) raised it by 72% (40 min). Since NE raised plasma tyrosine by stimulating alpha-adrenoceptors, as shown by phenoxybenzamine (PB) completely abolishing this increase, an experiment was done to find out whether IMI lowered plasma tyrosine by blocking alpha-adrenoceptors. In contrast to PB, IMI pretreatment failed to alter the NE-induced elevation in plasma tyrosine, suggesting that at this dose IMI is not an effective alpha-adrenergic antagonist in vivo. Thus, IMI would not appear to reduce plasma tyrosine by blocking alpha-adrenoceptors. In a separate experiment, propranolol blocked the ability of IMI to lower plasma tyrosine. Propranolol also prevented a 17% elevation in brain tryptophan levels induced by IMI but did not alter the 29% decrease in plasma tryptophan. PB by itself decreased plasma tyrosine, but this decrease was not greater by additionally treating with IMI. Salbutamol (10 mg/kg), a beta 2 agonist, lowered plasma tyrosine to 76% and raised brain tryptophan to 143% of control. These results suggest that IMI decreases tyrosine concentrations in plasma and raises tryptophan in brain by stimulating beta-adrenoceptors.