Biotransformation of cortexolone to hydrocortisone by molds using a rapid color-development assay

The capacity of 22 molds for 11β-hydroxylation of cortexolone (Reichstein’s compound S) to hydrocortisone were assessed. The biotransformation capacity was compared for solid-state and submerged monocultures of molds that were otherwise under identical conditions. Thin-layer chromatography and a novel rapid color-development assay were used to qualitatively establish the ability of fungi to convert cortexolone to hydrocortisone. These assays were validated and supplemented with data from high-performance liquid chromatography to obtain quantitative information on biotransformation. Nearly all the fungi consumed a significant fraction of the cortexolone fed, but only four of them (i.e., two isolates of Cunninghamella blakesleeana, C. echinulata, and Curvularia lunata) yielded measurable quantities of hydrocortisone. Submerged cultures generally gave a significantly greater yield of hydrocortisone compared to equivalent solid-state cultures. The text was submitted by the authors in English.     

Glucocorticoids Increase Tyrosine Hydroxylase Activity in Cultured Murine Neuroblastoma

Cyclic AMP and glucocorticoids appear to have a role in regulating the activity of tyrosine hydroxylase (TH), as well as the expression of "morphological differentiation" in murine neuroblastoma. Monolayer cultures of C-1300 murine neuroblastoma (clone NBP2) were treated with the following compounds in ethanol: dexamethasone, triamcinolone acetonide, hydrocortisone, cortexolone, androstenedione, testosterone, estradiol-17 beta; or with the phosphodiesterase inhibitor Ro20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone]. Treatment with either 200 micrograms/ml Ro20-1724 or 50 micrograms/ml dexamethasone produced significant increases in TH activity compared to alcohol controls (1.44 vs. 0.82 nmol 14CO2/mg protein/hr compared to 0.095). Triamcinolone acetonide or hydrocortisone also produced smaller, but significant, increases in TH activity compared to dexamethasone. When steroid activities were compared at 25 microM concentration and after 60 min of incubation (to maximize TH activity), triamcinolone acetonide was not as effective (62%) as dexamethasone. The relatively inactive glucocorticoid cortexolone produced a slight but significant increase, while the androgens androstenedione and testosterone and the estrogen estradiol-17 beta were without effect.     

Poly-N-vinylcaprolactam gel: A novel matrix for entrapment of microorganisms

Summary A new gel-type support poly-N-vinylcaprolactam for microbial cell immobilization is presented. The method allows one to obtain beads of biocatalyst in a single step. The properties of beads obtained using different types of gel stabilizers were compared; the best stabilizer was found to be tannin. The method developed was used for entrapment of viable bacterial cells and fungal spores. The biocatalysts obtained were used for transformations of both hydrophilic (sorbitol, indolyl-3-acetic acid) and lipophilic (cortexolone, hydrocortisone) substrates.Abbreviations PVC poly-N-vinylcaprolactam - ImC immobilized cells - IAA indolyl-3-acetic acid - TLC thin layer chromatography     

Concurrent corticosteroid and phenanthrene transformation by filamentous fungus Cunninghamella elegans

A filamentous fungus Cunninghamella elegans IM 1785/21Gp which displays ability of 17alpha,21-dihydroxy-4-pregnene-3,20-dione (cortexolone) 11-hydroxylation (yielding epihydrocortisone (eF) and hydrocortisone (F)) and polycyclic aromatic hydrocarbons (PAHs) degradation, was used as a microbial eucaryotic model to study the relationships between mammalian steroid hydroxylation and PAHs metabolization. The obtained results showed faster transformation of phenanthrene in Sabouraud medium supplemented with steroid substrate (cortexolone). Simultaneously phenanthrene stimulated epihydrocortisone production from cortexolone. In phenanthrene presence the ratio between cortexolone hydroxylation products (hydrocortisone and epihydrocortisone) was changed from 1:5.1-6.2 to 1:7.6-8.4 in the culture without phenanthrene. Cytochrome P-450 content significantly increased after the culture supplementation by the second substrate, phenanthrene or cortexolone, adequately. To confirm the involvement of cytochrome P-450 in phenanthrene metabolism, the inhibition studies were performed. The cytochrome P-450 inhibitors SKF 525-A (1.5mM) and 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone) (2mM) inhibited phenanthrene transformation by 80 and 62%, respectively. 1-aminobenzotriazole (1mM) completely blocked phenanthrene metabolism. The obtained results suggest a presence of connections between steroid hydroxylases and enzymes involved in PAH degradation in C. elegans.     

Microbial transformation of hydrocortisone by two fungal species Fusarium fujikuroi PTCC 5144 and Rhizomucor pusillus PTCC 5134

Abstract

This paper examines the biotransformation of hydrocortisone (1) by Fusarium fujikuroi and Rhizomucor pusillus. These species have not previously been tested for hydrocortisone biotransformation. The metabolites produced during hydrocortisone biotransformation by these two fungi were 11β,17α,20β,21-tetrahydroxypregn-4-en-3-one (2) and 11 β-hydroxyandrost-4-en-3,17-dione (3). Chemical structures were determined by spectroscopic methods. A time course study revealed that the disappearance of hydrocortisone was accompanied by the formation of metabolites 2 and 3. Metabolite 2 was produced as the major metabolite with high yield but the transformation to metabolite 3 was considerably lower, as determined by HPLC.     

The expression of cytochrome P-450 and cytochrome P-450 reductase genes in the simultaneous transformation of corticosteroids and phenanthrene by Cunninghamella elegans

The expression of cytochrome P-450 and cytochrome P-450 reductase (CPR) genes in the conterminous biotransformation of corticosteroids and PAHs was studied in Cunninghamella elegans 1785/21Gp. We had previously used this strain as a microbial eucaryotic model for studying the relationship between mammalian steroid hydroxylation and the metabolization of PAHs. We reported that cytochrome P-450 reductase is involved in the biotransformaton of cortexolone and phenanthrene. RT-PCR and Northern blotting analyses indicated that the cytochrome P-450 and CPR genes appear to be inducible by both steroids and PAHs. The expression of the cytochrome P-450 gene was increased ninefold and the expression of the CPR gene increased 6.4-fold in cultures with cortexolone and/or phenanthrene in comparison with controls. We conclude that the increase in cytochrome P-450 gene expression was accompanied by an increase in cytochrome P-450 enzymatic activity levels.     

Retinoid suppression of transglutaminase activity and envelope competence in cultured human epidermal carcinoma cells

Growth of SCC-13 squamous carcinoma cultures in the presence of retinoids considerably reduced the expression of two differentiation markers, the cellular capability to form cross-linked envelopes, and the enzyme transglutaminase required for cross-linking. A limited survey of retinoids showed that all-trans retinoic acid, 13-cis retinoic acid, and arotinoid Ro 13-6298 were highly effective in the absence of hydrocortisone and were only slightly antagonized by its presence in the medium. In contrast, retinyl acetate, retinol, and retinol bound to its plasma binding protein were quite active in the absence of hydrocortisone but were essentially inactive in its presence. Dexamethasone was also highly effective in antagonizing the suppressive action of retinyl acetate on envelope formation, while the corticosteroid antagonists cortexolone and progesterone were inactive. These results suggest that there are separate pathways, which are differentially regulated by hydrocortisone, for either the metabolism or action of retinol and retinoic acid in SCC-13 cells.     

Air oxidation of 17-hydroxycorticosteroids catalyzed by cupric acetate: formation of hemiacetal dimers

Hydrocortisone, cortexolone, hydrocortisone-17-butyrate, and budesonide were oxidized into alpha-ketoaldehydes by air exposure in the presence of Cu(OAc)(2). When free hydroxyl functions were present at position 17, hydrocortisone and cortexolone, the formed oxidation products, were identified as hemiacetal dimeric structures involving the free hydroxyl functions at position 17 and the newly formed aldehydes at position 21. Dimeric structures were established by using 1H913C0 correlations (HSQC and HMBC) and 1H-1H correlations (COSY and ROESY). The hemiacetal function was further confirmed by reaction of the dimer formed from hydrocortisone with two equivalents of 3-methyl-2-benzotriazolinone hydrazine (MTBH), giving quantitatively two equivalents of the 3-methyl-2-benzotriazolinone hydrazone of 21-dehydrohydrocortisone. When no free hydroxyl function was present as in the case of hydrocortisone-17-butyrate and budesonide, the expected alpha-ketoaldehydes were obtained.     

Isolation of wood-inhabiting fungi from Canadian hardwood logs

Wood-inhabiting fungi include many molds, wood-staining fungi, and decay fungi. Most of these fungal species can result in economic losses to wood users. Studies on molds, staining fungi, and decay fungi are necessary to be able to control their growth on wood and wood products. In this study, wood-inhabiting fungi were isolated from logs of 3 major Canadian hardwood species: sugar maple, white birch, and yellow birch. Two media were used for isolation. From these 3 wood species, a total of 1198 fungal cultures were obtained from summer- and winter-harvested logs in dry storage and under water sprinkling. The results showed that most fungal species were not host specific and affected all of the wood species tested. Frequently isolated molds were Alternaria alternata, Trichoderma species, and Mucor/Rhizopus (Zygomycota) species, frequently isolated staining fungi were Ophiostoma piceae and Ophiostoma piliferum, a frequently isolated bark saprophyte was Nectria cinnabarina, and frequently isolated decay fungi were taxa of the phylum Basidiomycota. More fungal species were isolated from summer-harvested logs than from winter-harvested logs. Fewer fungal cultures, especially decay fungi, were isolated from logs in early storage than from logs in late storage.     

华根霉固态与液态培养产胞外蛋白的比较蛋白质组学分析

【目的】考察固态和液态两种培养方式对丝状真菌华根霉(Rhizopus chinensis)CCTCC M201021产胞外蛋白的影响,以加深对微生物固、液态培养特异性产酶的认识。【方法】利用成分相同的培养基对华根霉分别进行平板固态培养和液态培养,提取华根霉所产胞外蛋白,采用二维凝胶电泳(2-DE)结合质谱分析,分离鉴定差异蛋白。【结果】固态培养下华根霉所产胞外蛋白的蛋白酶活性是液态培养的9.2倍。胞外蛋白质组数据分析表明,华根霉固态和液态培养所产胞外蛋白存在显著差异,其中约70%是固态和液态培养下各自产生的特有蛋白。质谱鉴定进一步表明,固、液态培养对华根霉产胞外蛋白的种类和表达量都有显著影响,其中水解酶类所占比例较大,主要是与蛋白质降解相关的蛋白。【结论】固态和液态不同培养方式影响了华根霉产胞外蛋白的组成,有些基因只在特定培养方式下表达。固态培养下华根霉产胞外蛋白酶种类相对更多以及大部分蛋白酶的上调表达,可能是固态培养下胞外蛋白酶活性很高的原因。研究结果提示需要注意固液态不同培养方式下丝状真菌胞外酶的筛选和生产可能存在的不一致性。     

Solid-state and membrane-surface liquid cultures of micromycetes: Specific features of their development and enzyme production (a Review)

Specific features in the development of micromycetes, typical mechanisms of their enzyme production, and conditions providing for an increase in enzyme secretion by the microscopic fungi in solid-state (on natural substrates and inert carriers) and membrane-surface liquid cultures are considered. The prospects and advantages of these fermentation methods for the production of extracellular enzymes are discussed and compared with submerged cultures.     

两株真降解菜籽饼中植酸的研究

利用选择性培养基从土壤中分离到两株能降解植酸的丝状真菌。这些菌株能利用肌醇作为唯一的碳源和能源而生长。在液态发酵中植权的降解率分别为７４．４％和９５．０％;在固太发酵中植酸的降解率为４０％左右。某些金属离子对菌株的降解率的提高具有一定的促进作用。对温度、ｐＨ和水分等影响因了也进行了初步的探讨。经初步鉴定,这两株菌中有一株为拟青霉（Ｐａｅｃｉｌｏｍｙｃｅｓ ｓｐ）,另一株为青霉（Ｐｅｎｉｃｉｌｌｉｕ     

两株真菌降解菜籽饼中植酸的研究

利用选择性培养基从土壤中分离到两株能降解植酸的丝状真菌。这些菌株能利用肌醇作为唯一的碳源和能源而生长。在液态发酵中植酸的降解率分别为74．4％和95．0％;在固态发酵中植酸的降解率为40％左右。某些金属离子对菌株的降解率的提高具有一定的促进作用。对温度、pH和水分等影响因子也进行了初步的探讨。经初步鉴定,这两株菌株中有一株为拟青霉（Paecilomycessp）,另一株为青霉（Penicilliumsp．）,它们均不产黄曲霉素素。     

11beta-Hydroxylation of Cortexolone (Reichstein Compound S) to Hydrocortisone by Curvularia lunata Entrapped in Photo-Cross-Linked Resin Gels

Spores of Curvularia lunata were immobilized by entrapment with photo-cross-linkable resin prepolymers and incubated to form mycelium in potato dextrose broth containing cortexolone (Reichstein compound S) as an inducer of steroid 11beta-hydroxylase. In a buffer system containing 2.5% dimethyl sulfoxide, this immobilized mycelium hydroxylated cortexolone to hydrocortisone. The activity of this mycelium was comparable to the activity of free mycelium. Dimethyl sulfoxide did not inhibit hydroxylase activity at the concentration used and was effective in dissolving the product. Of the various photo-cross-linkable resin prepolymers examined, use of ENT-4000, whose main chain was polyethylene glycol 4000 (chain length, approximately 40 nm), resulted in maximum hydroxylation activity of the entrapped mycelium. The chain length of prepolymers affected markedly mycelial growth in the gels and, subsequently, the activity of the entrapped mycelium. The immobilized hydroxylation system was more stable than the system in free mycelium and could be reactivated by incubation of the entrapped mycelium in potato dextrose broth containing cortexolone. The system was tested 50 times during 100 days of operation and was found to carry out the desired transformation with overall yields of 60%.     

11β-Hydroxylation of Cortexolone (Reichstein Compound S) to Hydrocortisone by Curvularia lunata Entrapped in Photo-Cross-Linked Resin Gels

Spores of Curvularia lunata were immobilized by entrapment with photo-cross-linkable resin prepolymers and incubated to form mycelium in potato dextrose broth containing cortexolone (Reichstein compound S) as an inducer of steroid 11β-hydroxylase. In a buffer system containing 2.5% dimethyl sulfoxide, this immobilized mycelium hydroxylated cortexolone to hydrocortisone. The activity of this mycelium was comparable to the activity of free mycelium. Dimethyl sulfoxide did not inhibit hydroxylase activity at the concentration used and was effective in dissolving the product. Of the various photo-cross-linkable resin prepolymers examined, use of ENT-4000, whose main chain was polyethylene glycol 4000 (chain length, approximately 40 nm), resulted in maximum hydroxylation activity of the entrapped mycelium. The chain length of prepolymers affected markedly mycelial growth in the gels and, subsequently, the activity of the entrapped mycelium. The immobilized hydroxylation system was more stable than the system in free mycelium and could be reactivated by incubation of the entrapped mycelium in potato dextrose broth containing cortexolone. The system was tested 50 times during 100 days of operation and was found to carry out the desired transformation with overall yields of 60%.     

In vitro effects of different steroid hormones on superoxide anion production of human neutrophil granulocytes

Neutrophil granulocytes play an important role in atherogenesis also through their free radical generation. According to recent studies, a point of action by which estrogens can provide protection against atherosclerosis is their inhibiting effect on superoxide anion production. The aim of our study was to test whether this means a common effect of steroids on superoxide production, or whether various steroid hormones have different action on superoxide generation of human granulocytes. Neutrophils were separated from the blood samples of twelve healthy volunteers. Isolated cells were incubated with different concentrations (10(-9), 10(-8), 10(-7) M) of hydrocortisone, aldosterone, cortexolone, 17-beta-estradiol, progesterone, and testosterone. Superoxide anion production was determined by photometry using the reduction of ferricytochrome-C. Compared to that of control cells neutrophils incubated with 17-beta-estradiol, progesterone, testosterone and hydrocortisone showed significantly reduced superoxide production. No significant alteration of superoxide anion production was found after the incubation of cells with aldosterone and cortexolone. It is concluded that similarly to estradiol other sex steroids and cortisol can inhibit the free radical production of human granulocytes, but mineralocorticoid aldosterone and Reichstein's substance S do not show such activity. Our results provide new evidence supporting the theory that certain types of steroid hormones have antioxidant capacity. This may give further reasons for investigating the molecular background of the existence or absence of this property and thus might lead to the development of new free radical scavengers.     

Does cyclic-3′,5′-AMP induce glutamine synthetase in embryonic neural retina?

The inducibility of retinal glutamine synthetase (GS) by dibutyryl cyclic-3′,5′-AMP (DB-cAMP) was re-examined in view of conflicting reports. Various lots of DB-cAMP were compared for a) ability to induce GS in cultures of embryonic chick neural retina, and b) their composition as visualized by paper chromatography. Chromatographically purified DB-cAMP did not induce retinal GS, nor did cAMP, DB-cGMP, epinephrine, or norepinephrine; none of these enhanced the induction of GS by hydrocortisone. Some of the agents occasionally caused small increases in GS activity; however, these were invariably below the GS levels induced consistently by hydrocortisone. A single lot of DB-cAMP was found which significantly raised GS activity in the retina; it contained a contaminant which when isolated was found to be responsible for this effect.     

Biotransformation of 17α-ethynyl substituted steroidal drugs with microbial and plant cell cultures: A review

Structural modification of steroids through whole-cell biocatalysis is an invaluable procedure for the production of active pharmaceutical ingredients (APIs) and key intermediates. Modifications could be carried out with regio- and stereospecificity at positions hardly available for chemical agents. Much attention has been focused recently on the biotransformation of 17α-ethynyl substituted steroidal drugs using fungi, bacteria and plant cell cultures in order to obtained novel biologically active compounds with diverse structure features. Present article includes studies on biotransformation on 17α-ethynyl substituted steroidal drugs using microorganisms and plant cell cultures. Various experimental and structural elucidation methods used in biotransformational processes are also highlighted.     

Degradation of 4-fluorobiphenyl by mycorrhizal fungi as determined by (19)F nuclear magnetic resonance spectroscopy and (14)C radiolabelling analysis.

The pathways of biotransformation of 4-fluorobiphenyl (4FBP) by the ectomycorrhizal fungus Tylospora fibrilosa and several other mycorrhizal fungi were investigated by using (19)F nuclear magnetic resonance (NMR) spectroscopy in combination with (14)C radioisotope-detected high-performance liquid chromatography ((14)C-HPLC). Under the conditions used in this study T. fibrillosa and some other species degraded 4FBP. (14)C-HPLC profiles indicated that there were four major biotransformation products, whereas (19)F NMR showed that there were six major fluorine-containing products. We confirmed that 4-fluorobiphen-4'-ol and 4-fluorobiphen-3'-ol were two of the major products formed, but no other products were conclusively identified. There was no evidence for the expected biotransformation pathway (namely, meta cleavage of the less halogenated ring), as none of the expected products of this route were found. To the best of our knowledge, this is the first report describing intermediates formed during mycorrhizal degradation of halogenated biphenyls.