粗糙脉孢菌中甾醇还原酶基因erg24对麦角甾醇合成的影响

粗糙脉孢菌为子囊菌中的高效纤维素降解菌,可以直接以纤维素为营养源进行生长.本研究以粗糙脉孢菌为实验对象,利用基因工程技术构建甾醇还原酶基因erg24的高表达菌株,分别以蔗糖、麦麸、玉米秸秆、小麦秸秆、杨树木屑、水稻秸秆6种物质的粉末为碳源培养野生型粗糙脉孢菌和erg24高表达菌株,利用半定量RT-PCR测定在不同培养条...     

A convenient synthesis of labelled rhodopsin and studies on its active site

Digitonin solutions of labelled rhodopsin, containing (3)H in the retinyl moiety, were prepared by two related methods. Labelled rhodopsin was also prepared for the first time in cetyltrimethylammonium bromide and purified by column chromatography. It was shown that only certain rhodopsin preparations on denaturation in the dark and the reduction with sodium borohydride gave up to 60% of the radioactivity in a fraction characterized as N-retinylphosphatidylethanolamine. Such preparations also gave a lipid-linked retinyl moiety at the metarhodopsin-I stage, but, as expected, a protein-linked retinyl moiety at the metarhodopsin-II stage. Other preparations however, gave exclusively protein-bound radioactivity at the native-rhodopsin, metarhodopsin-I and metarhodopsin-II stages. It is therefore conceivable that the formation of N-retinylphosphatidylethanolamine is due to a non-enzymic reaction resulting from the transfer of the retinyl moiety from its native site to an amino group of a favourably oriented phospholipid molecule. The only firmly established aspect of the rhodopsin active site remains the demonstration in our previous work that at the metarhodopsin-II stage the retinyl moiety is linked to an in-amino group of lysine. On the basis of chemical reactivity it is argued that the light-induced conversion of rhodopsin into metarhodopsin II involves a profound conformational change resulting in the dislocation of the retinylideneiminium chromophore from a non-polar environment in rhodopsin to a polar environment in metarhodopsin II.     

First synthesis of ent-desmosterol and its conversion to ent-deuterocholesterol

We report the first synthesis of the unnatural enantiomer of desmosterol (ent-desmosterol). The sterol nucleus was constructed enantiospecifically, followed by stepwise addition of the side chain. Beginning with ent-androst-4-ene-3,17-dione, ent-desmosterol was synthesized in 13 steps and 20% yield. Protected ent-desmosterol was subjected to catalytic deuteration to afford ent-deuterocholesterol. Ent-desmosterol and ent-deuterocholesterol will be used to study the importance of sterol absolute configuration for sterol-lipid interactions in biophysical studies and in biological systems.     

The conversion of 24-ethylidene sterols into poriferasterol by the alga Ochromonas malhamensis

A convenient method is described for the preparation of fucosterol-[7-³H₂] and 28-isofucosterol-[7-³H₂]. Both of these 24-ethylidene sterols, as well as 5α-stigmasta-7,Z-24(28)-diene-3β-ol-[2,4-³H₄], were converted into the 24β-ethyl sterol, poriferasterol, by cultures of the chrysophyte alga Ochromonas malhamensis. However, fucosterol-[7-³H₂] was not so efficiently incorporated as the other two compounds thus indicating that the configuration of the 24-ethylidene group is of some importance. It is suggested that a 24-ethylidene sterol of the Z-configuration is produced in de novo poriferasterol synthesis and that a Δ^22,24(28)-diene may be an important subsequent intermediate.     

The transfer of hydrogen from C-24 to C-25 in ergosterol biosynthesis

1. A convenient synthesis of 3-hydroxytrisnorlanost-8-en-24-al and its conversion into [24-(3)H]lanosterol and [26,27-(14)C(2)]lanosterol is described. 2. A method for the efficient incorporation of lanosterol into ergosterol by the whole cells of Saccharomyces cerevisiae is also described. 3. It is shown that in the biosynthesis of ergosterol from doubly labelled lanosterol the C-24 hydrogen atom of lanosterol is retained in ergosterol. 4. On the basis of unambiguous degradations it is shown that the C-alkylation step in ergosterol biosynthesis is accompanied by the migration of a hydrogen atom from C-24 to C-25. 5. The mechanism for the biosynthesis of the ergosterol side chain is presented. 6. Mechanisms of other C-alkylation reactions are also discussed.     

The transport of cytidine into rat brain in vivo,and its conversion into cytidine metabolites

Double-labeled cytidine, with a³H/¹⁴C isotope ratio of 20.00, has been intraventricularly injected into the brain of young rats, and its fate followed up to 90 min from administration together with the time-course of labeling. The injected nucleoside enters the brain as an intact molecule and is immediately utilized without prior degradation. Cytidine is actively converted into uridine and CMP, the latter being then transformed by a stepwise mechanism into CDP and CTP, and finally into CDP-choline and CDP-ethanolamine. The results indicate that administered cytidine represents a compound likely to enter metabolic events, which lead to CDP-choline and CDP-ethanolamine synthesis, and presumably to phospholipid production.     

Nutrient stoichiometry of aquatic hyphomycetes: Interstrain variation and ergosterol conversion factors

Ecological stoichiometry is a powerful concept. Rarely, however, has it been applied to fungi, despite their pivotal role in ecosystems. In view of the paucity of stoichiometric data, we grew 16 fungal isolates from streams in liquid culture (C:N:P = 160:16:1) and analysed them for nitrogen (N), phosphorus (P) and ergosterol as a fungal biomass marker. Interspecific differences explained up to 60% of the variation in N, P and ergosterol concentrations, and variation between strains of the same species accounted for up to another 16%. We found an average C:N:P of 136:10:1 in mycelia, while N:ergosterol and P:ergosterol ratios were 9.5 and 2.5, respectively. These ratios are an important step towards establishing reliable conversion factors to estimate the contribution of fungi to litter nutrient contents in complex field samples. Estimates could be further improved by applying the species-specific conversion factors we obtained. Additional analyses of fungal strains in conditions reflecting field situations are needed to strengthen the basis of such estimates of fungal nutrient pools in ecosystems; however, inherent variation within species limits the accuracy and precision that can be achieved.     

Apparent lack of conversion of sitosterol into C24-bile acids in humans

The metabolic fate of intravenously administered [4-14C]sitosterol was studied in two healthy subjects. In marked contrast to the results of a previous investigation with [22,23-3H]sitosterol, no detectable labeled C24-bile acid products appeared in bile. The first and rate-limiting step in the conversion of cholesterol into bile acids is catalyzed by the liver microsomal cholesterol 7 alpha-hydroxylase. When incubated with human liver microsomes, no detectable 7 alpha-hydroxylation of sitosterol could be demonstrated. This was the case also when using liver microsomes from two subjects treated with cholestyramine, in which case the rate of 7 alpha-hydroxylation of cholesterol was increased three- to sixfold. In order to bypass the rate-limiting step, the metabolic fate of 3H-labeled 7 alpha-hydroxysitosterol was studied in two volunteers. In this case there was a significant conversion into acid products in bile (18-32% excreted in bile during the first 17 h). Although part of the labeled products had chromatographic properties similar to those of cholic acid and chenodeoxycholic acid, further analysis showed that none of the products was identical to chenodeoxycholic acid and only traces at the most could be identical to cholic acid. The results suggest that healthy human subjects, in similarity with other mammalian species studied, have little or no capacity to convert sitosterol into the normal C24-bile acids.     

Facile preparation of deoxyiodocellulose and its conversion into 5,6-cellulosene

Treatment of 6-chloro-6-deoxycellulose with sodium iodide in 2,5-hexanedione gave 6-deoxy-6-iodocellulose; >80% of the chlorine atoms were replaced. 6-Deoxy-6-iodo-d-glucose was identified by g.l.c.-m.s. in hydrolyzates of deoxyiodocellulose. Acetylated 6-deoxy-6-iodocellulose was converted almost quantitatively into 5,6-cellulosene acetate, which was characterized by hydrolyzing the product and converting the resultant dicarbonyl sugar into an isopropylidene acetal 10. The changes of molecular-weight distribution during iodination and dehydroiodination were investigated by gel-permeation chromatography.     

Isolation of ergosterol peroxide and its reversion to ergosterol in the pathogenic fungus Sporothrix schenckii

Ergosterol peroxide, a presumed product of the H_{_2}O_{_2}-dependent enzymatic oxidation of ergosterol, has been isolated from yeast forms of the pathogenic fungus Sporothrix schenckii. The substance, which may have a role in fungal virulence, has been characterized mainly using spectroscopic methods (¹H and ¹³C nuclear magnetic resonance and high resolution mass spectra). The purified compound showed a molecular formula of C_{_28}H_{_44}O_{_3}, displaying characteristic features of epidioxy sterols and was reverted to ergosterol when submitted to S. schenckii enzymatic extract. This revised version was published online in June 2006 with corrections to the Cover Date.