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.     

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.     

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.     

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.     

24β-Ethyl-31-norlanosta-8,25(27)-dien-3β-ol and 24β-ethyl-25(27)-dehydrolophenol in seeds of three cucurbitaceae species

The structures of two 4α-methylsterols is isolated from Cucumis sativus(Cucurbitaceae) seeds were determined based mainly on their ¹³CNMR spectra as 24β-ethyl-31-norlanosta-8,25(27)-dien-3β-ol and 24β-ethyl-25(27)- dehydrolophenol, respectively, of which the former is a new sterol from natural sources. These two 4α-methylsterols were identified in the seeds of two other Cucurbitaceae species, Lagenaria leucantha var. Gourda and Citrullus battich. The probable biogenetic significance of the two 4α-methylsterols is discussed. Other 4α-methylsterols identified in the seeds of the three Cucurbitaceae species were obtusifoliol, cycloeucalenol and gramisterol.     

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.     

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.     

Mechanism of alkylation at C-24 during ergosterol biosynthesis in Phycomyces blakesleeanus

Ergosterol isolated from Phycomyces blakesleeanus grown in the presence of methionine-[methyl-²H₃] contained two ²H atoms showing that one ²H atom is lost during transmethylation. Ergosterol isolated from P. blakesleeanus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁] had a ¹⁴C:³H atomic ratio of 5:3. Chemical degradation of 2,3-dimethylbutanal obtained by ozonolysis of the doubly-labelled ergosterol showed that the ³H atom originally at C-24 of lanosterol is transferred to C-25 of ergosterol during transmethylation. The mechanism of formation of the ergosterol side chain in P. blakesleeanus is presented.     

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.     

Configuration at C-24 of sterols from the marine phanerogames Posidonia oceanica and Cymodocea nodosa

Sterols were extracted from two marine phanerogames, Posidonia oceanica and Cymodocea nodosa. The two plants contain 24α-ethyl sterols, while the 24α-methyl sterols are accompanied by 24β-epimers. The most abundant components are sitosterol, cholesterol and stigmasterol.     

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.     

Configuration at C-24 of sterols from the liverwort Palavicinnia lyellii

The 4-desmethylsterol fraction of the liverwort Palavicinnia lyellii is composed of 36% 24β-methylcholest-5-en-3β-ol (dihydrobrassicasterol), 16% 24α-methylcholest-5-en-3β-ol (campesterol), 33% 24α-ethylcholest-5-en-3β-ol (sitosterol) and 15% 24ξ-ethylcholesta-5,22-dien-3β-ol.     

Short hybrid peptides incorporating β- and γ-amino acids as antimicrobial agents

The peptides containing β- and γ-amino acids, LA-Lys(Z)-PEA, P1; LA-Lys(Z)-β^3,3-Ac₆c-PEA, P2; LA-Orn(Z)-β^3,3-Ac₆c-PEA, P3; LA-Lys(Z)-Gpn-PEA, P4; LA-Orn(Z)-Gpn-PEA, P5; LA-Lys(Z)-γ⁴-Phe-PEA, P6, LA-γ⁴-Leu-Lys(Z)-PEA, P7 and LA-β^3,3-Pip(Ac)-Lys(Z)-PEA, P8 were synthesized, characterized and evaluated against Gram-positive and Gram-negative bacteria. Among all, peptides P2, P3, P4 and P5 exhibited potent activity (MIC 6.25 μM) against S. aureus MTCC 737 and P. aeruginosa MTCC 424. In order to understand the efficacy of peptides and mechanism of action, time kill kinetics and fluorescence microscopic studies were performed against S. aureus and P. aeruginosa for the peptides P2, P3, P4 and P5. P4 took half time to show the bactericidal effect on P. aeruginosa and S. aureus in comparison to P2 at their 2x MICs. Fluorescence microscopic studies suggested that peptides P2 and P4 both killed the bacteria via membrane disruption. Further, P4 exhibited lowest haemolytic activity among active peptides and negligible cytotoxic activity against human cancer cell lines A-549, PC-3 and HCT-116 at its MIC.     

Lithocarpic acids O–S,five homo-cycloartane derivatives from the cupules of Lithocarpus polystachyus

Chemical investigation of the cupules of Lithocarpus polystachyus resulted in the identification of four 3,4-seco-homo-cycloartane and one homo-cycloartane derivatives named lithocarpic acids O–S. Their structures were determined based on extensive 1D/2D NMR, IR, MS spectroscopic analyses and chemical methods. Lithocarpic acid O (1) exhibited inhibitory activities on mouse and human isozymes of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) with IC₅₀ values of 0.49 and 1.1 μM, respectively.     

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.     

Multi-functional glycoside hydrolase: Blon_0625 from Bifidobacterium longum subsp. infantis ATCC 15697

We here describe a unique β-D-glucosidase (BGL; Blon_0625) derived from Bifidobacterium longum subsp. infantis ATCC 15697. The Blon_0625 gene was expressed by recombinant Escherichia coli. Purified recombinant Blon_0625 retains hydrolyzing activity against both p-nitrophenyl-β-D-glucopyranoside (pNPG; 17.3 ± 0.24 U mg⁻¹) and p-nitrophenyl-β-D-xylopyranoside (pNPX; 16.7 ± 0.32 U mg⁻¹) at pH 6.0, 30 °C. To best of our knowledge, no previously described BGL retains the same level of both pNPGase and pNPXase activity. Furthermore, Blon_0625 also retains the activity against 4-nitrophenyl-α-l-arabinofranoside (pNPAf; 5.6 ± 0.09 U mg⁻¹). In addition, the results of the degradation of phosphoric acid swollen cellulose (PASC) or xylan using endoglucanase from Thermobifida fusca YX (Tfu_0901) or xylanase from Kitasatospora setae KM-6054 (KSE_59480) show that Blon_0625 acts as a BGL and as a β-D-xylosidase (XYL) for hydrolyzing oligosaccharides. These results clearly indicate that Blon_0625 is a multi-functional glycoside hydrolase which retains the activity of BGL, XYL, and also α-l-arabinofuranosidase. Therefore, the utilization of multi-functional Blon_0625 may contribute to facilitating the efficient degradation of lignocellulosic materials and help enhance bioconversion processes.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

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.     

The conversion of 5α-lanost-24-ene-3β,9α-diol and parkeol into poriferasterol by the alga Ochromon as malhamensis

The 4,4-dimethylsterols 4α-lanost-24-ene-3β,9α-diol-[2-³H₂] and parkeol-[2-³H₂] were synthesized from lanosterol and subsequently incubated with cultures of Ochromonas malhamensis. 5α-Lanost-24-ene-3β,9α-diol was converted into poriferasterol with three times the efficiency of parkeol. Clionasterol was also found to be labelled from both parkeol and 5α-lanost-24-ene-3β,9α-diol. No significant incorporation of radioactivity into sterols was obtained after feeding 5α-lanost-24-ene-3β,9α-diol to higher plants, the chlorophyte alga Trebouxia, yeast or a cell free homogenate of rat liver.     

Synthesis and evaluation of 6-(3-[18F]fluoro-2-hydroxypropyl)-substituted 2-pyridylbenzothiophenes and 2-pyridylbenzothiazoles as potential PET tracers for imaging Aβ plaques

3-[¹⁸F]Fluoro-2-hydroxypropyl substituted compounds were synthesized and evaluated as novel ¹⁸F-labeled PET tracers for imaging Aβ plaque in a living brain. All compounds exhibited high binding affinities toward the synthetic Aβ_1–42 aggregate and/or Alzheimer’s disease brain homogenate. In the microPET study with normal mice, the 3-[¹⁸F]fluoro-2-hydroxypropyl substituted compounds resulted in fast brain washout by reducing the lipophilicities of the compounds. Intriguingly, (S)-configured PET tracers, (S)-[¹⁸F]1b and (S)-[¹⁸F]1c, exhibited a 2.8 and 4.0-fold faster brain washout rate at a peak/30 min in the mouse brain than the corresponding (R)-configured PET tracers despite there being no meaningful difference in binding affinities toward Aβ plaque. A further evaluation of (S)-[¹⁸F]1c with healthy rhesus monkeys also revealed excellent clearance from the frontal cortex with ratios of 7.0, 16.0, 30.0 and 49.0 at a peak/30, 60, 90, and 120 min, respectively. These results suggest that (S)-[¹⁸F]1c may be a potential PET tracer for imaging Aβ plaque in a living brain.