Biotransformation of progesterone by suspension cultures of Digitalis purpurea cultured cells

It has been shown that the cultured cells of Digitalis purpruea are capable of transforming progesterone (I) to 5α-pregnane-3,20-dione (II), 5α-pregnan-3β-ol-20-one (III), its glucoside (IV), 5α-pregnane-3β,20α-diol (V), its glucoside (VI), 5α-pregnane-3β,20β-diol (VII), its glucoside (VIII), Δ⁴-pregnen-20α-ol-3-one (IX), its glucoside (X), Δ-pregnen-20β-ol-3-one (XI) and its glucoside (XII). 5α-Pregnan-3β-ol-20-one glucoside (IV), 5α-pregnane-3β,20α-diol glucoside (VI), 5α-pregnane-3β,20β-diol glucoside (VIII), Δ⁴-pregnen-20α-ol-3-one glucoside (X) and Δ⁴-pregnen-20β-ol-3-one glucoside (XII) have been found for the first time as new metabolises by plant tissue cultures. A scheme for the biotransformation of progesterone (I) has been proposed, and the reduction and glucosidation activities distinctly have been observed in these cultured cells.     

Steroidal constituents of Solanum xanthocarpum

From the extract of the fruits of Solanum xanthocarpum, cycloartanol (I), cycloartenol (II), sitosterol (III), stigmasterol (IV), campesterol (V), cholesterol (VI), sitosteryl glucoside (VII), stigmasteryl glucoside (VIII), solamargine (IX), and β-solamargine (X) were identified and an isolated steroid (XI) was identical with 4α-methyl-(24R)-ethylcholest-7-en-3β-ol synthesized from carpesterol.     

Chemical Studies on Ambergris

Dihydro-α-ionol (II) was converted to dihydro-α-ionyl bromide (III) and dihydro-α-ionyl tosylate (V), which afforded their Wittig reagents (IV), (VI) on heating with triphenylphosphine. The Wittig reaction of ambreinolal-tetrahydropyranylether (VII) with the above Wittig reagents (IV) or (VI) gave α-ambrein-tetrahydropyranylether (VIII).     

Sterols from Candida lipolytica yeast grown on n-alkanes

The sterols of Candida lipolytica grown on n-alkanes were isolated by reverse phase HPLC and found to be mainly ergosterol, with small quantities of ergost-7-en-3β-ol, ergosta-7,22-dien-3β-ol, ergosta-7,24(28)-dien-3β-ol and ergosta-5,7,9(11),22-tetraen-3β-ol.     

Two new sterols from Chlorella ellipsoidea

Eight sterols were observed in Chlorella ellipsoidea and the four major components were identified as ergosterol, 5α-ergost-7-en-3β-ol, 22-trans-ergosta-5,8(9),22-trien-3β-ol and ergosta-5,8(9)-dien-3β-ol. This is the first report of the latter two sterols from green plants.     

The steroids and fatty acids of the basidiomycete Scleroderma polyrhizum

The fruit bodies of the Basidiomycete Scleroderma polyrhizum have been shown to contain the steroids ergosta-4,6,8(14) 22-tetraen-3-one and 5α,8α-epidoxyergosta-6,22-dien-3β-ol and also palmitic and oleic acids.     

Infection of Keratinocytes with Trichophytum rubrum Induces Epidermal Growth Factor-Dependent RNase 7 and Human Beta-Defensin-3 Expression

Human keratinocytes are able to express various antimicrobial peptides (AMP) to protect the skin from exaggerated microbial colonization and infection. Recently, in vitro growth-inhibiting activity of the skin-derived AMP psoriasin, RNase 7 and human beta-defensin (hBD)-2 against dermatophytes such as Trichophyton (T.) rubrum have been reported. To evaluate whether keratinocytes are able to respond to T. rubrum infection by an induced expression of AMP we exposed primary keratinocytes to living conidia of T. rubrum. This led to conidia germination and mycelial growth which was paralleled by a strong gene induction of the skin-derived AMP RNase 7 and hBD-3. Gene expression of the AMP psoriasin (S100A7) and hBD-2 were only slightly induced. The T. rubrum-mediated RNase 7 gene induction was accompanied by increased secretion of RNase 7. Parallel treatment of the keratinocytes with T. rubrum and the cytokine combination IL-17A/IFN-γ resulted in synergistic induction of RNase 7 and hBD-3 expression. Since patients receiving therapy by inhibition of the epidermal growth factor receptor (EGFR) more often suffer from dermatophytoses we investigated whether EGFR may be involved in the T. rubrum-mediated RNase 7 and hBD-3 induction. Primary keratinocytes incubated with an EGFR blocking antibody as well as with the EGFR antagonist AG1478 showed a significantly diminished RNase 7 and hBD-3 induction upon exposure of the keratinocytes to T. rubrum indicating that EGFR is involved in the T. rubrum-mediated induction of RNase 7 and hBD-3. The growth of T. rubrum in vitro was inhibited by hBD-3 in a dose-dependent manner suggesting that hBD-3 may contribute to cutaneous innate defense against T. rubrum. Taken together our data indicate that keratinocytes are able to initiate a fast defense response towards T. rubrum by the increased expression of AMP active against T. rubrum. A dysregulation of AMP may contribute to chronic and recurring dermatophytoses.     

Effect of ay-9944 on sterol biosynthesis in Chlorella sorokiniana

When Chlorella sorokiniana was grown in the presence of 4 ppm AY-9944 total sterol production was unaltered in comparison to control cultures. However, inhibition of sterol biosynthesis was shown by the accumulation of a number of sterols which were considered to be intermediates in sterol biosynthesis. The sterols which were found in treated cultures were identified as cyclolaudenol, 4α,14α-dimethyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 4α,14α-dimethyl -5α-ergosta-8,25-dien-3β-ol, 14α-methyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 24-methylpollinastanol, 14α-methyl-5α-ergost-8-en-3β-ol, 5α-ergost -8(14)-enol, 5α-ergost-8-enol, 5α-ergosta-8(14),22-dienol, 5α-ergosta-8,22-dienol, 5α-ergosta-8,14-dienol, and 5α-ergosta-7,22-dienol, in addition to the normally occurring sterols which are ergosterol, 5α-ergost-7-enol, and ergosta-5,7-dienol.The occurrence of these sterols in the treated culture indicates that AY-9944 is an effective inhibitor of the Δ⁸ → Δ⁷ isomerase and Δ¹⁴-reductase, and also inhibits introduction of the Δ²²-double bond. The occurrence of 14α-dimethyl-5α-ergosta-8,25-dien-3β-ol and 14α-methyl-9β,19-cyclo-5α-ergost -25-en-3β-ol is reported for the first time in living organisms. The presence of 25-methylene sterols suggests that they, and not 24-methylene derivatives, are intermediates in the biosynthesis of sterols in C. sorokiniana.     

Triterpenoid saponins from Astragalus wiedemannianus Fischer

Three cycloartane-type triterpene glycosides were isolated from Astragalus wiedemannianus together with eight known secondary metabolites namely cycloastragenol, cycloascauloside B, astragaloside IV, astragaloside VIII, brachyoside B, astragaloside II, astrachrysoside A, and astrasieversianin X. The structures were established mainly by a combination of 1D and 2D-NMR techniques as 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl]-25-O-β-D-glucopyranosyl-20(R),24(S)-epoxy-3β,6α,16β,25-tetrahydroxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-24-O-α-(4’-O-acetoxy)-L-arabinopyranosyl-16-O-acetoxy-3β,6α,16β,24(S),25-pentahydroxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-24-O-α-L-arabinopyranosyl-16-O-acetoxy-3β,6α,16β,24(S),25-pentahydroxycycloartane. To the best of our knowledge, the presence of an arabinose moiety on the acyclic side chain of cycloartanes is reported for the first time.     

Incorporation of methionine-[methyl-2H3] and mevalonic acid-[2-14C,(4R)-4-3H1] into Phycomyces blakesleeanus sterols

Ergosterol, episterol, 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol and 24-methylene-24,25-dihydrolanosterol, isolated from Phycomyces blakesleeanus grown in the presence of methionine-[methyl-²H₃], each contained two deuterium atoms; lanosterol, however, was unlabelled. The ¹⁴C:³H atomic ratio of the following sterols isolated from P. blakesleeanus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁], was: ergosterol, 5:3; episterol, 5:4; ergosta-5,7,24(28)-trien-3β-ol, 5:3; 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol, 5:4; 24-methylene-24,25-dihydrolanosterol, 6:5; lanosterol, 6:5. The significance of these results in terms of ergosterol biosynthesis is discussed.     

Synthesis of homoursodeoxycholic acid and [11,12-3H]homoursodeoxycholic acid

Homoursodeoxycholic acid and [11,12-³H]homoursodeoxycholic acid were synthesized from ursodeoxycholic acid and homocholic acid, respectively. Ursodeoxycholic acid (Ia) was converted to 3α,7β-diformoxy-5β-cholan-24-oic acid (Ib) using formic acid. Reaction of the diformoxy derivative (Ib) with thionyl chloride yielded the acid chloride (II) which was treated with diazomethane to produce 3α,7β-diformoxy-25-diazo-25-homo-5β-cholan-24-one (III). Homoursodeoxycholic acid (IV) was formed from the diazoketone (III) by means of the Wolff rearrangement of the Arndt-Eistert synthesis.N-Bromosuccinimide oxidation of homocholic acid (V), which was prepared from cholic acid by the same procedure described above, afforded 3α,12α-dihydroxy-7-oxo-25-homo-5β-cholan-25-oic acid (VI). Reduction of the 7-ketohomodeoxycholic acid (VI) with sodium in 1-propanol gave 3α,7β,12α-trihydroxy-25-homo-5β-cholan-25-oic acid (VII). The methyl ester of 7-epihomocholic acid (VII) was partially acetylated to give methyl 3α,7β-diacetoxy-12α-hydroxy-25-homo-5β-cholan-25-oate (VIII) using a mixture of acetic anhydride, pyridine and benzene. Dehydration of the diacetoxy derivative (VIII) with phosphorus oxychloride yielded methyl 3α,7β-diacetoxy-25-homo-5β-chol-11-en-25-oate (IX). Reduction of the unsaturated ester (IX) with tritium gas in the presence of platinum oxide catalyst followed by alkaline hydrolysis gave [11,12-³H]homoursodeoxycholic acid.     

Total lipid and sterol components of Rhizopus arrhizus: identification and metabolism

The total lipid levels and the incorporation of [¹⁴C]acetate during the life cycle of the fungus Rhizopus arrhizus were investigated. The total lipid abundances ranged between 2.2 and 15.3% of the tissue, reaching the maximum midway through the 6-day growth period. The sporangiospores contained 2.65% total lipids.The sterols were also investigated and ranged in concentration from 1.88 to 9.1% of the total lipids during the growth period. 4,4-Dimethyl, 4α-methyl, and 4-des-methyl sterols were tentatively identified by tlc and the latter group separated and identified by combined glc-ms. The predominant 4-desmethyl sterols were ergosta-Δ^5,7,22-trienol (ergosterol), ergosta-Δ^7,22-dienol (5-dihydroergosterol), ergost-Δ⁷-enol (fungisterol), and the tentatively identified ergosta-Δ^5,7,14-trienol in relative concentrations of 24.6, 58.8, 9.9, and 6.7%, respectively. The sterol components of the spores were qualitatively identical to those of the mycelial tissues, but several minor components remain to be identified.     

Detection of the ergosterol and episterol isomers lichesterol and fecosterol in nystatin-resistant mutants of Neurospora crassa

Wild-type Neurospora crassa is completely inhibited by 5 ppm nystatin. Ultraviolet-induced mutants have been isolated that grow in the presence of 60 ppm of the antibiotic. Gas-liquid chromatographic, mass spectroscopic, and nuclear magnetic resonance analyses showed the wild-type sterols to be ergosterol (ergosta-5,7,22-trien-3β-ol) and episterol (ergosta-7,24(28)-dien-3β-ol) in a 3:1 ratio. The mutants contained lichesterol (ergosta-5,8,22-trien-3β-ol) and fecosterol (ergosta-8,24(28)-dien-3β-ol) in a 2:1 ratio, differing from the wild type only in the position of the B-ring unsaturation. A deficiency of an ergosta-8,24 (28)-dien-3β-ol:ergosta-7,24(28)-dien-3β-ol isomerase is indicated.     

Increase in Resistance to Fluconazole and Itraconazole in Trichophyton rubrum Clinical Isolates by Sequential Passages In Vitro under Drug Pressure

Trichophyton rubrum, an anthropophilic dermatophyte fungus, is the predominant causative agent of superficial skin infections in human population. There are only scanty reports on drug susceptibility profiling of T. rubrum. Neither mechanisms for drug resistance development nor correlation between in vitro drug susceptibility and in vivo response to treatment is known for that species. In this study, changes in the in vitro susceptibilities to fluconazole (FLZ) and itraconazole (ITZ) among thirty T. rubrum clinical strains subjected to sequential passages in the presence or absence of the azoles were investigated. Each strain was passaged 12 times at 4-week intervals as three parallel cultures, maintained on a drug-free medium (1), and a medium containing FLZ (2) or ITZ (3) at subinhibitory concentrations. Susceptibility to FLZ and ITZ of the original strain and its 3 subcultures was determined by microdilution method. The MIC values of the two azoles remained unaltered for all T. rubrum strains tested, after 12 passages on a drug-free medium. Among the strains grown with FLZ, an increase in the MICs of FLZ and ITZ was noted in 17 (56.7 %) and 19 (63.3 %) strains, respectively. Increased MICs of ITZ and FLZ were demonstrated for 24 (80 %) and 20 (66.7 %) strains that were propagated with ITZ. The results indicate the capacity of T. rubrum to develop resistance toward the azoles after prolonged exposure to these drugs. Resistance of T. rubrum to azoles plays an important role in therapy failures and consequently contributes to persistence and chronicity of the infections.     

Formation of a 5 hydroxy sterol by Saccharomyces cerevisiae

The incorporation of [28 ¹⁴C] ergosta-7,24(28)-dien-3β-ol into ergosta-7,22-dien-3β,5α-diol by aerobically growing $\text{S.}\text{cerevisiae}$ has established its presence in this organism. This, coupled with previous work, is considered to be substantive evidence for the operation of a hydroxylation-dehydration mechanism in the introduction of Δ⁵ unsaturation in ergosterol biosynthesis in yeast.     

Synthesis of Some New α- and β-(l→2) Linked Disaccharides Containing L-Quinovose (6-Deoxy L-Glucose)

Hepta-O-acetyl-2-0-β-l-quinovopyranosyl-α-d-glucose (VI) and hepta-O-acetyl-2-O-α-l-quinovopyranosyl-β-d-gIucose (VIII) were prepared by the coupling of 2,3,4-tri-O-acetyl-α-l-quinovopyranosyl bromide (IV) with l,3,4,6-tetra-O-acetyl-α-D-glucose (V) in the presence of mercuric cyanide and mercuric bromide in absolute acetonitrile.

Similarly, hepta-O-acetyW-O-α-l-quinovopyranosyl-α-d-galactose (X) and hepta-O-acetyl-2-O-β-L-quinovopyranosyl-α-d-galactose (XI) were prepared by the reaction of IV with 1,3,4,6-tetra-O-acetyl-α-d-galactose (IX).

Removal of the protecting groups of VI, VIII, X and XI afforded the corresponding disaccharides. On treatment with hydrogen bromide, VI, VIII, X and XI gave the corresponding acetobromo derivatives.     

Biosynthesis of Δ5.23 sterols in etiolated coleoptiles from Zea mays

In addition to the previously found ergosta-5, E-23-dien-3β-ol and 5α-ergosta-7, E-23-dien-3β-ol, the following Δ23 sterols have been identified in etiolated maize coleoptiles: cyclosadol, 4α, 14α-dimethyl-5α-ergosta-8, E-23-dien-3β-ol, 4α, 14α-dimethyl-9β, 19-cyclo-5α-ergosta-8, E-23-dien-3β-ol and 4α-methyl-5α-ergosta-7, E-23-dien-3β-ol. The incubation of maize coleoptile microsomes in the presence of cycloartenol and of [¹⁴C-methyl]S-adenosyl methionine gave a mixture of labelled 24-methylene cycloartanol and cyclosadol. No trace of cyclolaudenol could be detected in these conditions. It is suggested that Δ²³ sterols are products of the C-24 methyltransferase reaction and they probably do not arise from a Δ²⁴ → Δ²³ isomerization occurring at a later stage of the biosynthesis. The Δ¹³-sterols may play an intermediary role in the biosynthesis of 24-methyl sterols in this plant material.     

Sterols of Xanthoria parietina: Evidence for two sterol ‘pools’ and the identification of a novel C,8 triene,ergosta-5,8,22-trien-3β-ol

Sterols extracted from Xanthoria parietina with organic solvents and released by saponification of the residual lichen tissue were analysed by GC-MS. The main components of the solvent-extractable sterols were two C₂₈ trienes and those of the more tightly bound sterols were ergost-5-en-3β-ol and two C₂₉ compounds. The structures of the C₂₈ compounds were shown to be ergosta-5,7,22-trien-3β-ol, Ia (ergosterol) and the previously unreported ergosta-5,8,22-trien-3β-ol, IIa, for which the name lichesterol is proposed. The main C₂₉ sterol was identified as (24R)-24-ethylcholesta-5,22-dien-3β-ol (poriferasterol).     

Arylglycerol-γ-Formyl Ester as an Aromatic Ring Cleavage Product of Nonphenolic β-O-4 Lignin Substructure Model Compounds Degraded by Coriolus versicolor

4-Ethoxy-3-methoxyphenylglycerol-γ-formyl ester (compound IV) was identified as a degradation product of both 4-ethoxy-3-methoxyphenylglycerol-β-syringaldehyde ether (compound I) and 4-ethoxy-3-methoxyphenylglycerol-β-2,6-dimethoxyphenyl ether (compound II) by a ligninolytic culture of Coriolus versicolor. An isotopic experiment with a ¹³C-labeled compound (compound II′) indicated that the formyl group of compound IV was derived from the β-phenoxyl group of β-O-4 dimer as an aromatic ring cleavage fragment. However, compound IV was not formed from 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether (compound III). γ-Formyl arylglycerol (compound IV) could be a precursor of 4-ethoxy-3-methoxyphenylglycerol (compound VI), because 3-(4-ethoxy-3-methoxyphenyl)-1-formyloxy propane (compound VII) was cleaved to give 3-(4-ethoxy-3-methoxyphenyl)-1-propanol (compound VIII) by C. versicolor. 4-Ethoxy-3-methoxyphenylglycerol-β,γ-cyclic carbonate (compound V), previously found as a degradation product of compound III by Phanerochaete chrysosporium (T. Umezawa, and T. Higuchi, FEBS Lett., 25:123-126, 1985), was also identified from the cultures with compound I, II, and III and degraded to give the arylglycerol (compound VI). An isotopic experiment with ¹³C-labeled compounds II′ and III′ indicated that the carbonate carbon of compound V was derived from the β-phenoxyl groups of β-O-4 substructure.     

Monocyte-Derived Dendritic Cells from Patients with Dermatophytosis Restrict the Growth of Trichophyton rubrum and Induce CD4-T Cell Activation

Dermatophytes are the most common agents of superficial mycoses that are caused by mold fungi. Trichophyton rubrum is the most common pathogen causing dermatophytosis. The immunology of dermatophytosis is currently poorly understood. Recently, our group investigated the interaction of T. rubrum conidia with peritoneal mouse macrophages. We found that macrophages phagocytose T. rubrum conidia resulted in a down-modulation of class II major histocompatibility complex (MHC) antigens and in the expression of co-stimulatory molecules. Furthermore, it induced the production of IL-10, and T. rubrum conidia differentiated into hyphae that grew and killed the macrophages after 8 hrs of culture. This work demonstrated that dendritic cells (DCs) and macrophages, from patients or normal individuals, avidly interact with pathogenic fungus T. rubrum. The dermatophyte has two major receptors on human monocyte-derived DC: DC-SIGN and mannose receptor. In contrast macrophage has only mannose receptor that participates in the phagocytosis or bound process. Another striking aspect of this study is that unlike macrophages that permit rapid growth of T. rubrum, human DC inhibited the growth and induces Th activation. The ability of DC from patients to interact and kill T. rubrum and to present Ags to T cells suggests that DC may play an important role in the host response to T. rubrum infection by coordinating the development of cellular immune response.