The effect of cerulenin on sterol biosynthesis inSaccharomyces cerevisiae

Cerulenin specifically inhibited fatty acid biosynthesis inSaccharomyces cerevisiae without having an effect on sterol formation. Ergosterol was not required for cell growth in the presence of cerulenin (1 μg/ml). The addition of fatty acids to the growth medium reduced the amount of ergosterol formed by 45%; further addition of cerulenin to the media had no effect on the amount of ergosterol synthesized by the cells. The incorporation of³H from³H₂O into ergosterol was not affected by cerulenin whereas incorporration into fatty acids was inhibited by 90%.     

General resistance to sterol biosynthesis inhibitors inSaccharomyces cerevisiae

Screening for resistance to fenpropimorph was undertaken in order to isolate yeast mutants affected in the regulation of the ergosterol pathway. Among the mutants isolated, one bearing the recessivefen1-1 mutation was characterized by a 1.5-fold increase in the ergosterol level and a general resistance to sterol biosynthesis inhibitors. Thefen1-1 mutation was linked toMAT locus on chromosome III. The measurement of enzyme activities involved in the ergosterol pathway revealed that isopentenyl diphosphate (IPP) isomerase activity was specifically increased 1.5-fold as compared to the wild type strain. However, overexpression of IPP isomerase in the wild type strain was not by itself sufficient to lead to sterol increase or resistance to sterol biosynthesis inhibitors, showing that IPP isomerase is not a limiting step in the pathway. Thefen1-1 mutation permits viability in aerobiosis of yeast disrupted for sterol-14 reductase in absence of exogenous ergosterol supplementation, whereas the corresponding strain bearing the wild typeFEN1 allele grows only in anaerobiosis. This result shows that ignosterol is able to efficiently replace ergosterol as bulk membrane component and that thefen1-1 mutation eliminates the specific ergosterol requirement in yeast.     

Regulation of the sequencing in sterol biosynthesis

The evolutionary development and sequencing of events in the biosynthetic pathway leading to sterols is reviewed and illustrated by recent experiments from the author's laboratory. One of 12 papers being published from the “Sterol Symposium” presented at the AOCS Meeting, New Orleans, April 1970.     

Developmental regulation of sterol biosynthesis inCucurbita maxima L.

Twenty-two sterols were identified by capillary gas chromatography and capillary gas chromatography/mass spectroscopy inCucurbita maxima grown under green-house conditions. Both whole plants and individual tissues (leaves, stems, roots, cotyledons, flowers) were analyzed at weekly intervals during the 12-week development of the plant. In whole plants, sterol accumulation parallels plant growth except for a period in the mid-life cycle where there is a reduction in the amount of sterol accumulated on a total sterol/plant and mg sterol/g dry wt basis. This reduction in the amount of sterol is coincident with the visual onset of flowering. During development, the percent contribution of each class of sterol (Δ^5_, Δ^7_, Δ^0_-sterols) remains relatively constant. However, the percent contribution of an individual sterol species varies depending on the tissue examined and the developmental period selected for analysis. While the young plant (<2 weeks) possesses elevated levels of sterols with the Δ²⁵⁽²⁷⁾-double bond, the trend was toward a reduction in the amounts of these sterols with development. Leaves and stems accumulate large quantities of 24ζ-ethyl-5α-cholesta-7,22-dien-3β-ol (7,22-stigmastadienol) and 24ζ-ethyl-5α-cholest-7-en-3β-ol (7-stigmastenol), while roots accumulate only 7,22-stigmastadienol as their principal sterol. Male flowers and roots were found to contain elevated levels of Δ^5_-sterols.     

The steric requirements for sterol inhibition of tetrahymanol biosynthesis

Many naturally occurring sterols are accumulated and metabolized byTetrahymena pyriformis. In most cases, the sterols are desaturated to give^Δ5,7,22-derivatives. Compounds with an ethyl, but not with a methyl, substituent at C-24 are dealkylated. Exposure of the ciliates to the appropriate sterol sharply curtails the synthesis of the native pentacyclic triterpenoid alcohols, tetrahymanol and diplopterol. An analysis with modified sterols has revealed several additional features that are required for desaturation at C-7,8 and C-22,23 and for inhibition of tetrahymanol biosynthesis. The presence of atrans-17(20)-double bond, which eliminates free rotation at C-20 and fixes C-22 to the right of the nucleus, does not interfere with desaturation, while thecis- or left-handed isomer is not metabolized. Thecis-Δ¹⁷⁽²⁰⁾-isomer is, however, an effective inhibitor of tetrahymanol biosynthesis, although less so that thetrans-counterpart. When a methyl or hydroxyl group at C-20 protrudes to the front of the molecule in the right-handed conformation, metabolism is reduced or abolished. Shortening (by one C-atom) or lengthening of the sterol side chain has little effect on the ability of the compounds to inhibit tetrahymanol biosynthesis or to support growth, as long as the overall length of the side chain does not exceed seven carbons from C-20. The presence of a 7α-, 7β-, 20α-, 20β-, or a 25-hydroxy group in the cholesterol molecule sharply inhibits desaturation and curtails the effectiveness of the compound as an inhibitor of tetrahymanol biosynthesis. The 7- or 22-keto derivatives seem to act in a fashion similar to the hydroxy derivatives, but these compounds show greater inhibition of growth. 20-Methylcholesterol, however, is a potent inhibitor of synthesis, which suggests that the polarity of the substituent of C-20 is more important than bulk. Many sterols can effectively replace tetrahymanol in the membranes of these ciliates. However, several of the compounds, which inhibit synthesis, appear to be physiologically inappropriate, and poor growth results. An example of the latter class is 20-methylcholesterol. Finally, a class of sterols, represented by 20α-hydroxycholesterol and 7-ketocholesterol, does not severly inhibit tetrahymanol synthesis but leads to growth inhibition and surface abnormalities. These sterols apparently lead to a disordered membrane, even in the presence of tetrahymanol.     

Absence of sterol biosynthesis in oyster tissue culture

An oyster heart tissue culture was used to investigate the ability of the American oyster (Crassostrea virginica) to synthesize sterols from [¹⁴C]acetate. Lipids were extracted from cultured oyster heart cells and no label was found in the purified fatty acid fraction, 90% of which was located in the saturated and monounsaturated fractions. Although these results cannot rule out the possibility of sterol synthesis in other oyster tissues, this and previous evidence are in support of the hypothesis that the American oyster is unable to synthesize sterols.     

Developmental regulation of sterol biosynthesis inZea mays

Sixty-one sterols and pentacyclic triterpenes have been isolated and characterized by chromatographic and spectral methods fromZea mays (corn). Several plant parts were examined; seed, pollen, cultured hypocotyl cells, roots, coleoptiles (sheaths), and blades. By studying reaction pathways and mechanisms on plants fed radiotracers ([2-¹⁴C]mevalonic acid, [2-¹⁴C]acetate, and [2-³H]acetate), and stable isotopes (D₂O), we discovered that hydroxymethylglutaryl CoA reductase is not “the” rate-limiting enzyme of sitosterol production. Additionally, we observed an ontogenetic shift and kinetic isotope effect in sterol biosynthesis that was associated with the C-24 alkylation of the sterol side chain. Blades synthesized mainly 24α-ethyl-sterols, sheaths synthesized mainly 24-methyl-sterols, pollen possessed an interrupted sterol pathway, accumulating 24(28)-methylene-sterols, and germinating seeds were found to lack an activede novo pathway. Shoots, normally synthesizing (Z)-24(28)-ethylidine-cholesterol, after incubation with deuterated water, synthesized the rearranged double-bond isomer, stigmasta-5,23-dien-3β-ol. Examination of the mass spectrum and¹H nuclear magnetic resonance spectrum of the deuterated 24-ethyl-sterol indicated the Bloch-Cornforth route originating with acetyl-CoA and passing through mevalonic acid to sterol was not operative at this stage of development. An alternate pathway giving rise to sterols is proposed. Dedicated to the distinguished insect steroid biochemist Dr. James A. Svoboda on occasion of his sixtieth birthday. Based on a paper presented at the Symposium on the “Regulation of Biosynthesis and Function of Isopentenoids”, Atlanta, Georgia, May 1994.     

Involvement of heme components in sterol metabolism ofSaccharomyces cerevisiae

There is an intimate association between sterol biosynthesis in yeast and aerobicity. Besides the requirement for molecular oxygen for the epoxidation of squalene, cytochrome hemoproteins are involved in demethylation and desaturation steps. Regulatory effects of hemes on sterol formation have been demonstrated using specifically defective mutants of yeast. Heme competency participates in a mechanism whereby wild-type cells are prevented from taking exogenous sterols from the growth media. The multiple interactions of hemes and sterols appear to be associated with the variously defined functions for sterols in the yeast cells. Based on a paper presented at the Symposium on Plant and Fungal Sterols: Biosynthesis, Metabolism and Function, held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Effects of triarimol and tridemorph on sterol biosynthesis inSaprolegnia ferax

The effects of two fungicides, triarimol and tridemorph, on sterol biosynthesis inSaprolegnia ferax were examined. Cultures grown in the presence of triarimol accumulated lanosterol. Tridemorph-treated cultures accumulated Δ⁸-sterols including zymosterol, fecosterol and stigmasta-8-24(28)-dienol. The latter is a new sterol. A proposed scheme is given for sterol biosynthesis inS. ferax showing points of inhibition of triarimol and tridemorph. Results point to the intermediacy of lanosterol but not cycloartenol in sterol synthesis inSaprolegnia.     

AY-9944 inhibition of sterol biosynthesis inChlorella emersonii

WhenChlorella emersonii, a green alga, was cultured in the presence of 20 ppm AY-9944, a number of sterols accumulated which appear to be intermediates of sterol biosynthesis in this organism. The sterols isolated include 14α-methyl-ergost-8-en-3β-ol, 14α-methyl 24S-stigmast-8-en-3β-ol, 14α-methyl ergosta-8,24(28)-dien-3β-ol and 4α, 14α-dimethyl 24S-stigmast-8-en-3β-ol. Smaller quantities of several other sterols were found in addition to the normally occurring Δ⁷, chondrillasterol and Δ⁷. Control cultures were found to contain, in addition to the normally occurring sterols, smaller quantities of most of the sterols isolated from AY-9944 inhibited cultures. AY-9944 is a specific inhibitor of Δ⁷ in cholesterol biosynthesis in animals. However, sinceC. emersonii terminates sterol biosynthesis one step prior to the Δ⁷ step, AY-9944 apparently inhibits sterol biosynthesis prior to this step in this organism. The accumulation of 14α-methyl sterols in treated cultures suggests that AY-9944 is an effective inhibitor of the 14α-methyl removal inC. emersonii. Scientific Article No. A1865, Contribution No. 4775 of the Maryland Agricultural Experiment Station.     

Aspects of sterol metabolism in the yeastSaccharomyces cerevisiae and inPhytophthora

Using 5 wild-type strains of yeast, nonequivalence in the isolation of sterol mutants was observed. Experiments are described on the effects of sterol modifications on growth, physical and enzymic properties ofSaccharomyces cerevisiae andPhytophthora cactorum. Although discontinuities in Arrhenius kinetics were observed by fluorescence anisotropy and enzymic measurements of mutants (but not wild-types) of yeast, evidence based on membrane permeability and differential scanning calorimetry failed to support bulk lipid phase transitions as the cause for the discontinuities.     

Inhibition of sterol biosynthesis in chlorella ellipsoidea by AY-99441

WhenChlorella ellipsoidea was grown in the presence of 4 ppm AY-9944, complete inhibition of Δ⁵-sterol biosynthesis was achieved. However total sterol production remained unaltered. As a result a number of sterols accumulated that appear to be intermediates in sterol biosynthesis. These sterols were described and identified as (24S)-5α-ergost-8(9)-en3β-ol, (24S)-5α-stigmast-8(9)-en-3β-ol, 4α-methyl-(24S)-5α-ergosta-8, 14-dien-3β-ol, 4α-methyl-(24S)-5α-stigmasta-8, 14-dien-3β-ol, 4α-methyl-(24S)-5α-ergost-8(9)-en-3β-ol and (24S)-4α-methyl-5α-stigmast-8(9)-en-3β-ol. The occurrence of these sterols inChlorella ellipsoidea is the first time they have been noted in biological material. The accumulation of these sterols in treated cultures indicates that AY-9944 is an extremely effective inhibitor of the Δ⁸→Δ⁷ isomerase and the Δ¹⁴ reductase of these plants. The occurrence of small amounts of other sterols in treated cultures has led to a proposed pathway for thebiosynthesis of sterols inChlorella ellipsoidea. Scientific Article No. A1775, Contribution No. 4565 of the Maryland Agricultural Experiment Station.     

The Roles of Monomeric GTP-Binding Proteins in Macroautophagy in Saccharomyces cerevisiae

Autophagy is a cellular degradation process that sequesters components into a double-membrane structure called the autophagosome, which then fuses with the lysosome or vacuole for hydrolysis and recycling of building blocks. Bulk phase autophagy, also known as macroautophagy, controlled by specific Atg proteins, can be triggered by a variety of stresses, including starvation. Because autophagy relies extensively on membrane traffic to form the membranous structures, factors that control membrane traffic are essential for autophagy. Among these factors, the monomeric GTP-binding proteins that cycle between active and inactive conformations form an important group. In this review, we summarize the functions of the monomeric GTP-binding proteins in autophagy, especially with reference to experiments in Saccharomyces cerevisiae.     

Effects of triparanol and AY-9944 upon sterol biosynthesis inChlorella

The growth rates of three species ofChlorella were inhibited by triparanol and AY-9944.Chlorella emersonii was noticeably more resistant to both inhibitors than the other species. A large number of sterols were isolated and identified in inhibited cultures. Ca. 18 of these were identified from nature for the first time. Triparanol resulted in inhibition of the removal of the 14α methyl group. It also inhibited the second alkylation of the side chain and, in one species, strongly inhibited the Δ⁸ → Δ⁷ isomerase reaction. InC. ellipsoidea, triparanol also inhibited Δ¹⁴-reductase and Δ⁷-reductase. The introduction of the Δ²² double bond was inhibited by both drugs. The effect of AY-9944 was similar to that of triparanol inC. emersonii, but it was an extremely effective Δ¹⁴-reductase inhibitor inC. ellipsoidea. These various types of inhibition of sterol synthesis indicate a lack of specificity of both drugs inChlorella and suggest that primitive plants such as these may be valuable as test organisms in an evaluation of the activity of potential inhibitors of sterol biosynthesis.     

溶胶-凝胶法制备羟基磷灰石粉体

前言羟基磷灰石[Ca10（PO4）6（OH）2]（简称HA）化学组成接近生物体骨质的矿物成分,具有良好的生物活性及生物相容性,可以用来修复损坏或者病变的硬组织,在整形外科及口腔修复方面得到了广泛的应用。人们探索了多种方法合成羟基磷灰石粉体,如固相反应法、化学沉淀法、水热合成法以及溶胶-凝胶（sol—gel）法等。     

Expression of human asparagine synthetase in Saccharomyces cerevisiae

Human asparagine synthetase was expressed in the yeast Saccharomycescerevisiae. The identity of the expressed protein was confirmedby immunoblotting and in vitro enzymatic activity. The recombinantenzyme was shown to have both the ammonia-and glutamine-dependentasparagine synthetase activity in vitro. In contrast to overproductionin Escherichia coli, the expressed protein was found to be solublein the yeast cell. Furthermore, expression in yeast made itpossible to isolate non-degraded human asparagine synthetasewhich had also the N-terminal methionine correctly processed.The yeast expression plasmid was constructed for optimal productionof the recombinant enzyme. In addition, unique restriction enzymesites that bracket the first five codons of the human asparaginesynthetase gene were introduced. This will allow the use ofoligonucleotide cassette mutagenesis to investigate the roleof the N-terminal amino acids in asparagine synthetase enzymaticactivity.     

Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model

The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic models. The secretory pathway was the first trafficking pathway clearly understood mainly thanks to the work done in the laboratory of Randy Schekman in the 1980s. They have isolated yeast sec mutants unable to secrete an extracellular enzyme and these SEC genes were identified as encoding key effectors of the secretory machinery. For this work, the 2013 Nobel Prize in Physiology and Medicine has been awarded to Randy Schekman; the prize is shared with James Rothman and Thomas Südhof. Here, we present the different trafficking pathways of yeast S. cerevisiae. At the Golgi apparatus newly synthesized proteins are sorted between those transported to the plasma membrane (PM), or the external medium, via the exocytosis or secretory pathway (SEC), and those targeted to the vacuole either through endosomes (vacuolar protein sorting or VPS pathway) or directly (alkaline phosphatase or ALP pathway). Plasma membrane proteins can be internalized by endocytosis (END) and transported to endosomes where they are sorted between those targeted for vacuolar degradation and those redirected to the Golgi (recycling or RCY pathway). Studies in yeast S. cerevisiae allowed the identification of most of the known effectors, protein complexes, and trafficking pathways in eukaryotic cells, and most of them are conserved among eukaryotes.     

酿酒酵母菌对重金属的生物吸附作用研究进展

结合最近的研究成果,总结了酿酒酵母菌作为生物吸附材料的优点、表现形式和吸附性能,重点讨论了酿酒酵母菌的生物吸附机理,提出今后的研究方向。     

Design of high energy intermediate analogues to study sterol biosynthesis in higher plants

Several enzymes of plant sterol biosynthesis involve during their catalysis postulated or demonstrated carbocationic high energy intermediates (HEI). The aim of this study was to interfere with plant sterol biosynthesis by means of rationally designed species able to mimic these carbocationic HEI. It has been demonstrated previously that the design of transition state (TS) or HEI analogues could lead to powerful and specific inhibitors of enzymes. We applied this approach to the following target enzymes: 2,3-epoxy-2,3-dihydroqualene cyclase, AdoMet-cycloartenol-C-24-methyltransferase (AdoMet CMT), cycloeucalenol-obtusifoliol isomerase (COI) and Δ⁸-Δ⁷-sterol isomerase. Very potent inhibitors have been obtained in the four cases. As an example, analogues of cycloartenol substituted at C-25 by a charged heteroatom (N, As, S) have been synthesized and shown to be able to mimic the C-25 carbocationic HEI involved in the reaction catalyzed by the AdoMet CMT. These compounds were shown to be very potent and specific inhibitors of this enzyme both in vitro (Ki=2.10⁻⁸ M, Ki/Km=10⁻³) and in vivo. The potent inhibitors described are powerful tools to control in vivo the sterol profile of plant cells and therefore to study the structural and functional roles of sterols in cell membranes. Moreover, these compounds constitute leader molecules of a new class of rationally designed inhibitors which could be of value in plant protection.