Evidence for separate intermediates in the biosynthesis of 24α- and 24β-alkylsterols in tracheophytes

When mevalonate-[2-¹⁴C] was incubated with seeds of Pinus pinea, 23% of the label in sterols was found in trans-24-ethylidenecholesterol, 12% in a mixture of 24α- and 24β-methylcholesterol, and 65% in 24α-ethylcholesterol. However, when the radioactive substrate was lanosterol-[24-³H], label appeared only in the 24-ethylidene- (85%) and the epimeric 24-methylsterols (15%). From the ratios of labels in the ethylidene- and methyl-sterols it was possible to show that the tritium in the 24-C₁ -mixture was incorporated only into the 24β-methyl epimer. The labelling patterns are consistent with a pathway to 24β-alkylsterols via Δ²⁵⁽²⁷⁾-sterols bypassing 24-ethylidenesterols and to 24α-alkylsterols via Δ²⁴⁽²⁸⁾-sterols which are isomerized to Δ²⁴⁽²⁵⁾-sterols prior to reduction.     

Observations on the biosynthesis of 24-methylcholesterol and 24-ethylcholesterol by Zea mays

Examination of the sterols of Zea mays shoots has established that the 24-ethylcholesterol is predominately the 24α-epimer, sitosterol, but the 24-methylcholesterol is a mixture of the 24α- and 24β-epimers. After incubation of Z. mays shoots with [2-¹⁴C, (4R)4-³H₁]mevalonic acid the sitosterol had a ³H: ¹⁴C atomic ratio of 2.09:5 which is consistent with previous results indicating that a Δ²⁴⁽²⁵⁾ -sterol is implicated in its biosynthesis. By contrast, the 24α- and 24β-methylcholesterol mixture had a higher ³H: ¹⁴C atomic ratio of 2.82:5. This can be explained by the operation of two routes for the elaboration of the 24-methylcholesterol side chain. One may proceed via Δ²⁴⁽²⁵⁾- and Δ²⁴⁽²⁵⁾-sterols to produce the 24α-methylcholesterol with a ³H: ¹⁴C atomic ratio of 2:5. The other route may involve reduction of either a Δ²⁴⁽²⁸⁾-, a Δ²³- or a Δ²⁵-sterol intermediate to give the 24β1-methylcholesterol with a ³H: ¹⁴C atomic ratio of 3:5. The proportion of these two labelled compounds in the mixture then determines the observed ³H: ¹⁴C atomic ratio (2.82:5). Some evidence for the formation of a Δ²⁵-compound, cyclolaudenol, by Z. mays shoots was provided by incorporation studies employing either [2-¹⁴C]mevalonic acid or [Me-¹⁴C]methionine as the sterol precursor.     

Investigations on the Δ23-, Δ24(28)- and Δ25-Sterols of zea mays

The sterols of Zea mays shoots were isolated and characterized by TLC, HPLC, GC/MS and ¹H NMR techniques. In all, 22 4-demethyl sterols were identified and they included trace amounts of the Δ²³-, Δ²⁴- and Δ²⁵-sterols, 24-methylcholesta-5,E-23-dien-3β-ol, 24-methylcholesta-5,Z-23-dien-3β-ol, 24-methylcholesta-5,25-dien-3β-ol, 24-ethylcholesta-5,25-dien-3β-ol and 24-ethylcholesta-5,24-dien-3β-ol. In the 4,4-dimethyl sterol fraction, cycloartenol and 24-methylenecycloartanol were the major sterol components but small amounts of the Δ²³-compound, cyclosadol, and the Δ²⁵-compound, cyclolaudenol, were recognized. These various Δ²³- and Δ²⁵-sterols may have some importance in alternative biosynthetic routes to the major sterols, particularly the 24β-methylcholest-5-en-3β-ol component of the C₂₈-sterols. Radioactivity from both [2-¹⁴C]MVA and [methyl-¹⁴C]methionine was incorporated by Z. mays shoots into the sterol mixture. Although 24-methylene and 24-ethylidene sterols were relatively highly labelled, the various Δ²³- and Δ²⁵-sterols contained much lower levels of radioactivity, which is possibly indicative of their participation in alternative sterol biosynthetic routes. (24R)-24-Ethylcholest-5-en-3β-ol (sitosterol) had a significantly higher specific activity than the 24-methylcholest-5-en-3β-ol indicating that the former is synthesized at a faster rate.     

Influence of brassinosteroids on initiation of the root gravitropic response in Pisum sativum seedlings

In roots of Pisum sativum seedlings, the average lag-time required for initiation of the gravitropic response was reduced proportionally to the concentration of 24-epibrassinolide (EBL) added to the root solution (range of 10⁻¹³ to 10⁻⁸ M concentrations). A treatment with clotrimazole, a compound inhibiting steroid synthesis, prevents initiation of the gravitropic response. This effect was partly reverted by addition of EBL. From analysis of variability in the populations, it is suggested that BR conditions the root curvature through a gravitropic-induced change in sensitivity to the PGRs regulating cell elongation.     

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.     

Occurrence of 24-epimers of cycloart-25-ene-3β,24-diols in the stems of Euphorbia trigona

¹³C NMR spectroscopy has demonstrated that the cycloart-25-ene-3β,24-diol isolated from the stems of Euphorbia trigona is a 1:1 mixture of the 24-epimers. This seems to be the first instance of the detection of the natural occurrence of 24-epimeric cycloart-25-ene-3β,24-diols.     

The mechanism of alkylation at C-24 during clionasterol biosynthesis in Monodus subterraneus

Clionasterol isolated from Monodus subterraneus grown in the presence of methionine-[methyl-²H₃] contained four ²H atoms showing the participation of a 24-ethylidene sterol intermediate in its biosynthesis. Clionasterol isolated from M. subterraneus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁ had a ¹⁴C:₃H atomic ratio of 5:3 indicating that the 24-ethylidene sterol intermediate is reduced directly to clionasterol and not isomerized to a Δ²⁴-sterol which is then reduced.     

Metabolism of 24-dihydrolanosterol in Ochromonas malhamensis and Chlorella ellipsoidea

24-Dihydrolanosterol-[2-³H] was converted to cholesterol in Chlorella ellipsoidea but ergost-5-enol, poriferasterol, clionasterol were not labelled. The absence of the necessary 24(25) double bond precursor eliminates the possibility of C₂₈ and C₂₉ sterol synthesis. However, it was confirmed that 24-dihydrolanosterol was metabolized by Ochromonas malhamensis to give cholesterol, brassicasterol, and poriferasterol.     

Biosynthesis of makisterone A and 20-hydroxyecdysone from labeled sterols by the honey bee,Apis mellifera

In an effort to determine the sterol precursor(s) of the 28-carbon ecdysteroid, makisterone A, honey bee pupae (13 days post-oviposition) were injected with radiolabeled sterols and subsequently examined for labeled ecdysteroids. High performance liquid chromatography of the pupal extracts revealed that [³H]campesterol was converted to a compound that behaved chromatographically identical to authentic makisterone A, and [¹⁴C]cholesterol was incorporated into a compound chromatographically like 20-hydroxyecdysone. No incorporation of either 24-[³H]methylenecholesterol or [¹⁴C]sitosterol into an ecdysteroid was observed. The neutral sterols of uninjected honey bee pupae contained 49.8% 24-methylenecholesterol on a relative percent basis and, with three other C²⁸ and C²⁹ sterols, accounted for over 99% of the total sterols present.     

Sterol biosynthesis via cycloartenol in Saprolegnia

The Oomycete Saprolegnia ferax incorporates ³H from both cycloartenol-[2-³H] and lanosterol-[2-³H] into its normal sterols cholesterol, fucosterol, desmosterol, and 24-methylenecholesterol. It is concluded that sterol biosynthesis in this organism is via cycloartenol and the taxonomic implications are discussed.     

Biosynthesis of steroidal withanolides in Withania somnifera

Administration of 24-methylene-cholesterol-[28-³H] to Withania somnifera, yielded [³H] radioactivity in the isolated withaferin A and withanolide D, whereas administered 24-(R,S)-methyl-cholesterol-[28-³H] was not incorporated into these compounds. 24-Methylene-cholesterol is, therefore, proposed as a sterol precursor of the withanolides. A novel procedure is described for the isolation of withanolides from W. somnifera. This method in conjunction with an improved procedure for administration of labelled sterols and mevalonolactone produces a greatly increased yield of labelled withanolides.     

Phenotypic characterization of mammosphere-forming cells from the human MA-11 breast carcinoma cell line

The phenotypic diversity of breast carcinoma may be explained by the existence of a sub-population of breast cancer cells, endowed with stem cell-like properties and gene expression profiles, able to differentiate along different pathways. A stem cell-like population of CD44⁺CD24^−/low breast cancer cells was originally identified using cells from metastatic pleural effusions of breast carcinoma patients. We have previously reported that upon in vitro culture as mammospheres under stem cell-like conditions, human MA-11 breast carcinoma cells acquired increased tumorigenicity and lost CD24 expression compared with the parental cell line. We now report that upon passage of MA-11 mammospheres into serum-supplemented cultures, CD24 expression was restored; the rapid increase in CD24 expression was consistent with up-regulation of the antigen, and not with in vitro selection of CD24⁺ cells. In tumors derived from subcutaneous injection of MA-11 mammospheres in athymic nude mice, 76.1 ± 9.7% of cells expressed CD24, vs. 0.5 ± 1% in MA-11 cells dissociated from mammospheres before injection. The tumorigenicity of sorted CD44⁺CD24⁻ and CD44⁺CD24^high MA-11 cells was equal. Single cell-sorted CD24⁻ and CD24^high MA-11 gave rise in vitro to cell populations with heterogeneous CD24 expression. Also, subcutaneous tumors derived from sorted CD24⁻ sub-populations and single-cell clones had levels of CD24 expression similar to the unsorted cells. To investigate whether the high expression of CD24 contributed to the tumorigenic potential of MA-11 cells, we silenced CD24 by shRNA. CD24 silencing (95%) resulted in no difference in tumorigenicity upon s.c. injection in athymic nude mice compared with mock-transduced MA-11 cells. Since CD24 silencing was maintained in vivo, our data suggest that the level of expression of CD24 is associated with but does not contribute to tumorigenicity. We then compared the molecular profile of the mammospheres with the adherent cell fraction. Gene expression profiling revealed that the increased tumorigenicity of MA-11 mammospheres was associated with changes in 10 signal transduction pathways, including MAP kinase, Notch and Wnt, and increased expression of aldehyde dehydrogenase, a cancer-initiating cell-associated marker. Our data demonstrate that (i) the level of CD24 expression is neither a stable feature of mammosphere-forming cells nor confers tumorigenic potential to MA-11 cells; (ii) cancer-initiating cell-enriched MA-11 mammospheres have activated specific signal transduction pathways, potential targets for anti-breast cancer therapy.     

24α-Methyl-5α-cholest-7-en-3β-ol from seed oil of Helianthus annuus

24-Methyl-5α-cholest-7-en-3β-ol (24-methyllathosterol) isolated from the seed oil of Helianthus annuus was shown to have 24α-configuration by ¹H NMR spectroscopy. The stereochemistry at C-24 of some other 24-alkylsterols isolated from this plant material also was determined.     

Studies on the C-24 configurations of Δ7-sterols in theseeds of Cucurbita maxima

Uncertainties surrounding the structures of the Δ⁷-sterols in the seeds of Cucurbita maxima have been resolved. Seven components were found by TLC, GLC, HPLC, mass spectrometry and ¹H NMR. They were 24β-ethyl-5α-cholesta-7,22,25(27)-trien-3β-ol, 24β-ethyl-5α-cholesta-7,25(27)-dien-3gb-ol, avenasterol, spinasterol, 24-dihydrospinasterol, 24ζ-methyllathosterol and 25(27)-dehydrofungisterol. The ¹H NMR spectra indicated that the sterols with an ethyl substituent at C-24 occurred in the absence of their C-24 epimers. This seems to be the first instance of the detection of 25(27)-dehydrofungisterol in a higher plant.     

利用噬菌体肽库筛选与HIV-1p24抗原结合的多肽

通过噬菌体展示技术筛选出与HIV-1p24抗原结合的多肽,为用多肽辅助p24抗原检测提供实验基础.以重组p24抗原为靶蛋白,对噬菌体随机七肽库进行三轮筛选,用EUSA鉴定第三轮筛选到的噬菌体克隆与p24重组抗原的结合能力,再对噬菌体克隆进行测序分析,同时研究了ELISA中噬菌体加入量及多种封闭剂对噬菌体特异性结合能力的...     

Codisterol and other Δ5-sterols in the seeds of Cucurbita maxima

A substantial amount (ca 18%) of the sterol found in the seeds of Cucurbita maxima had a Δ-bond and consisted of seven components. They were identified as 25(27)-dehydroporiferasterol, clerosterol, isofucosterol, stigmasterol, sitosterol, campesterol and codisterol. The C-24 configuration of each of the sterols was unequivocally established by a ¹H NMR spectral comparison with authentic standards. This is the first time codisterol has been found in a higher plant and also the first time the structures and configurations of the Δ⁵-sterols from a Cucurbitaceae species have been clearly characterized.     

Direct interaction,instrumental for signaling processes,between LacCer and Lyn in the lipid rafts of neutrophil-like cells

Lactosylceramide [LacCer; β-Gal-(1-4)-β-Glc-(1-1)-Cer] has been shown to contain very long fatty acids that specifically modulate neutrophil properties. The interactions between LacCer and proteins and their role in cell signaling processes were assessed by synthesizing two molecular species of azide-photoactivable tritium-labeled LacCer having acyl chains of different lengths. The lengths of the two acyl chains corresponded to those of a short/medium and very long fatty acid, comparable to the lengths of stearic and lignoceric acids, respectively. These derivatives, designated C18-[³H]LacCer-(N₃) and C24-[³H]LacCer-(N₃), were incorporated into the lipid rafts of plasma membranes of neutrophilic differentiated HL-60 (D-HL-60) cells. C24-[³H]LacCer-(N₃), but not C18-[³H]LacCer-(N₃), induced the phosphorylation of Lyn and promoted phagocytosis. Incorporation of C24-[³H]LacCer-(N₃) into plasma membranes, followed by illumination, resulted in the formation of several tritium-labeled LacCer-protein complexes, including the LacCer-Lyn complex, into plasma membrane lipid rafts. Administration of C18-[³H]LacCer-(N₃) to cells, however, did not result in the formation of the LacCer-Lyn complex. These results suggest that LacCer derivatives mimic the biological properties of natural LacCer species and can be utilized as tools to study LacCer-protein interactions, and confirm a specific direct interaction between LacCer species containing very long fatty acids, and Lyn protein, associated with the cytoplasmic layer via myristic/palmitic chains.     

油菜素内酯对盐渍下油菜幼苗生长的调控效应及其生理机制

为了探讨油菜素内酯对植物耐盐性的调控,以甘蓝型油菜"南盐油1号"为试验材料,研究了外源24-表油菜素内酯(24-EBL)对100、200 mmol/L Na Cl胁迫下油菜幼苗干重(DW)、相对含水量(RWC)、渗透调节能力(OAA)、叶片气体交换参数、气孔限制值(Ls)等的调节效应,还测定了不同器官的Na+、K+、Cl-含量,并计算各器官的K+/Na+和SK,Na。结果表明:(1)在不同浓度的盐胁迫下,油菜幼苗DW显著下降,胁迫下外源喷施10-12、10-10、10-8、10-6mol/L 24-EBL作用下,油菜植株干重均不同程度的上升,且植株干重都在10-10mol/L 24-EBL(EBL2)处理下达到最大值,分别比100、200 mmol/L Na Cl胁迫下增加29%和20%。与对照相比,非盐胁迫下外源喷施10-12、10-10、10-8、10-6mol/L 24-EBL,油菜幼苗植株干重与对照相比均无显著变化。(2)不同Na Cl浓度胁迫下,油菜叶片的RWC显著下降,外施EBL2可显著提高油菜叶片的RWC和OAA。(3)不同浓度Na Cl胁迫下,油菜幼苗叶片净光合速率(Pn)、气孔导度(Gs)、胞间CO2浓度(Ci)和蒸腾速率(Tr)均不同程度下降,而Ls显著上升,而外喷EBL2可不同程度的提高Pn、Gs、Ci、Tr,降低Ls。(4)与对照相比,Na Cl胁迫下油菜幼苗叶片、叶柄和根的Na+和Cl-含量均显著上升,Na Cl浓度愈高,Na+和Cl-含量上升愈显著。而K+含量均下降,外源EBL2可显著降低幼苗各器官的Na+和Cl-含量,对幼苗叶片K+含量没有影响,但提高了叶柄和根中的K+含量。上述表明,合适浓度的24-EBL外喷可明显提高油菜的耐盐水平,且不同浓度Na Cl胁迫下,最适24-EBL浓度均为10-10mol/L。主要是因为外源喷施24-EBL能显著改善离子稳态和渗透调节能力,从而改善盐胁迫下油菜幼苗的光合作用、水分状况,提高其耐盐性。而24-EBL对盐处理下油菜植株气孔限制的显著改善是其促进其光合、水分利用的重要原因,也是其对100 mmol/L Na Cl处理的油菜生长调控效果优于200 mmol/L Na Cl处理的重要原因之一。结果还显示,在叶片中,24-EBL外施可通过排Na+和Cl-来维持植株离子稳态,而对K+影响不大;在根、茎中可通过排Na+、排Cl-、吸K+维持稳态。     

Ergosterol biosynthesis in Mucor pusillus

Ergosterol, 22-dihydroergosterol, obtusifoliol and 24-methylene-24,25-dihydrolanosterol, isolated from Mucor pusillus grown in the presence of methionine-[methyl-²H₃], each contained two deuterium atoms; lanosterol, however, was unlabelled. Ergosterol and 22-dihydroergosterol, isolated from M. pusillus grown in the presence of mevalonic acid-[2-¹⁴C, (4R)-4-³H₁] had ¹⁴C:³H atomic ratios of 5:3. The significance of these results in terms of sterol biosynthesis in this organism in general and alkylation at C-24 in particular is discussed.