The occurrence of polyprenols in seeds and leaves of woody plants

The contents of the heterogenous group of polyisoprenoids was found about two orders of magnitude lower in seeds than the amount of polyprenols and/or their carboxylic esters accumulated during vegetation season in leaves. In contrast to leaves, no seeds were found containing more than 0.5 mg of these lipids per gram of dry tissue. Almost 50% had less than 0.01 mg/g - the amount which is the limit of detection by the procedure used in this work. In gymnosperms (10 representatives of Cupressaceae, Pinaceae and Taxaceae) the polyprenol spectra in seeds and in needles were similar. In angiosperms (25 representatives of 13 botanical families) the polyisoprenoid mixture in seeds resembled the minor, additional subfamily found in leaves.     

Search for polyprenols in leaves of evergreen and deciduous Ericaceae plants.

Various species and cultivars of Ericaceae family were checked for the presence of long-chain polyprenols in their leaves. In the genus Rhododendron no polyprenols were found in the ever-green species, while they were present in the deciduous type. The polyprenols were of chain-length of 14-20 isoprene residues and they occurred in the form of acetic acid esters. The polyprenol accumulation is discussed with respect to senescence of leaves.     

The occurrence of unique, long-chain polyprenols in the leaves of Potentilla species

Polyprenols are accumulated in the leaves of Potentilla anserina at concentration up to 0.3% fresh weight. They constitute a mixture of poly-cis fully unsaturated analogues of up to 29 isoprene units long. In this and other species of Potentilla the polyprenol mixture is composed of two families, one grouping the medium chain-length polyprenols (built up of about 20 isoprene units), and the second one, composed mainly of very long prenolgues from 24 to approx. 28 isoprene units. This is a first report on the occurrence of polyprenyl alcohols of this chain length in plant material and the first one on the presence of multiple polyprenol mixture in angiosperms. A useful modification of polyprenols preparation from plant material, based on solid phase extraction with hydrophobic gel Lipidex-5000 is described.     

Light conditions alter accumulation of long chain polyprenols in leaves of trees and shrubs throughout the vegetation season

In many plants belonging to angiosperms and gymnosperms the accumulation in leaves of long chain polyprenols and polyprenyl esters during growth in natural habitats depends on the light intensity. The amount of polyprenols in leaves is also positively correlated with the thickness of the leaf blade (SLA, specific leaf area). The polyprenol content of leaves shows seasonal changes with a maximum in autumn and a minimum in early summer with the difference between poorly and well illuminated plants persisting throughout the vegetation season.     

The occurrence and seasonal distribution of C50-C60-polyprenols and of C100-and similar long-chain polyprenols in leaves of plants

Large amounts of fully unsaturated, mainly-cis, higher isoprenoid alcohols consisting of 17-30 isoprene units were found in several plants of the Rosaceae family. They occur as mixtures of several prenologues with either C85 - or C100 - prenol dominating in the form of acetates. The highest level of these polyprenols (0.5-1.0% of wet weight) were found in Crataegus crus- galli , Cotonoaster lucida, Prunus serotina and Sorbus suecica (intermedia). Their content increased with increasing age of the leaves. The dynamics of this rise is different from that observed in the case of accumulation of free C50- C60 - prenols (up to 0.5% of wet weight) in leaves of various plant species.     

银杏叶中聚戊烯醇含量及其季节性变化

银杏 (ＧｉｎｋｇｏｂｉｌｏｂａＬ .)叶中聚戊烯醇 (ｐｏｌｙｐｒｅｎｏｌｓ)含量较高[1] ,聚戊烯醇在人体中是多萜醇的中间体[2 ] ,对细胞膜糖蛋白生物合成具有重要作用[3 ] ,用于多发性硬化症 (痛风 ,红斑狼疮等 )等免疫功能疾病、糖尿病、慢性肝炎及肿瘤病人化疗的辅助治疗     

The production of long chain polyunsaturated fatty acids in transgenic plants by reverse-engineering

The nutritional importance of long chain polyunsaturated fatty acids (LC-PUFAs) is now well-established, with these lipids playing roles in human development and the prevention of various diseases. In particular, n-3 polyunsaturated fatty acids are protective against cardiovascular disease (CVD) and risks associated with metabolic syndrome. In view of the decline in marine fish stocks, which represent the predominant natural reserves of n-3 long chain polyunsaturates, alternative sources are urgently required. One approach may be to express the LC-PUFA-biosynthetic pathway in transgenic plants. Recent progress in validating this approach has now emerged, demonstrating the feasibility of using transgenic plants to synthesise these important human nutrients.     

High-performance liquid chromatography analysis of molecular species of sphingolipid-related long chain bases and long chain base phosphates in Saccharomyces cerevisiae after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

The molecular species of dihydrosphingosines and phytosphingosines and their 1-phosphates with carbon chain lengths from 16 to 20 have been tagged with the fluorescent amino group reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. All these derivatives could be resolved by reversed phase HPLC on a C18 column. A convenient one-pot method is described whereby lipid extracts from strains of Saccharomyces cerevisiae containing carbon chain length homologs of sphingolipid long chain bases and their phosphorylated derivatives were directly reacted with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, ester lipids were deacylated, and the reaction mixtures were subjected to liquid chromatography. Five molecular species of both sphingolipid long chain bases and their phosphorylated derivatives are for the first time separated and analyzed. The procedure is quite sensitive, requiring only approximately 10(8) wild-type cells.     

The isolation of peptides by high-performance liquid chromatography using predicted elution positions

This paper describes the derivation and use of predictive retention coefficients for the reversed-phase high-performance liquid chromatography of peptides. The use of predicted elution positions in the isolation of peptides is illustrated by two examples where peptides, whose existence was postulated from cDNA sequence data, have been successfully isolated. The combination of the powerful chromatographic technology and the ability to predict the elution positions of peptides based on their composition provides a very potent method for the isolation of peptides from biological tissues.     

Semipreparative isolation of individual cyanobacterial heterocyst-type glycolipids by reverse-phase high-performance liquid chromatography.

Procedures are described for the quantitative, semi-preparative isolation of individual cyanobacterial heterocyst-type glycolipids (HGs) by reverse-phase HPLC (RP-HPLC) and the modifications to conventional techniques necessary to prevent significant HG losses during sample preparation. Total lipids are obtained from cultures of nitrogen-fixing cyanobacteria by triplicate extraction with 200 packed-cell volumes of chloroform/methanol (1/1, v/v), filtered, and redissolved in chloroform for loading onto a short disposable column of acid-washed silica. After removal of neutral lipids, pigments, and the majority of monogalactosyldiacylglycerol with chloroform and chloroform/acetone, HGs are eluted along with other complex lipids in methanol. The complex lipids are then fractionated by TLC and the HGs isolated as two classes of differing mobilities. The individual components of each class are then resolved by isocratic elution with methanol/water (91/9, v/v) from a C18 RP-HPLC column with refractive index detection. Samples of up to approximately 1.0 mg lipid can be completely separated and the major components isolated in high purity from a single run. Structural studies on the major HG of Nostoc azollae show it to be the glycosylated hexacosane-1,3,25-triol found by others in Anabaena cylindrica.     

Single polyprenol and dolichol isolation by semipreparative high-performance liquid chromatography technique

A new method of separation of single polyprenols (or dolichols) from a mixture of isoprenoid alcohols is described. Application of a high-performance liquid chromatography (HPLC) apparatus equipped with a semipreparative ODS column resulted in preparation of long-chain (dihydro)polyprenols of high purity (>95%).This approach substantially decreases the time scale of the conventional chromatographical preparative procedure. The method can be widely used in chemical and biochemical projects, where single polyprenols or dolichols are required.     

The rapid isolation of vacuoles from leaves of crassulacean Acid metabolism plants

A technique is presented for the isolation of vacuoles from Sedum telephium L. leaves. Leaf material is digested enzymically to produce protoplasts rapidly which are partially lysed by gentle osmotic shock and the inclusion of 5 millimolar ethyleneglycol-bis (β-aminoethyl ether)N,N′-tetraacetic acid in the wash medium. Vacuoles are isolated from the partially lysed protoplasts by brief centrifugation on a three-step Ficoll-400 gradient consisting of 5, 10, and 15% (w/v) Ficoll-400. A majority of the vacuoles accumulate at the 5 to 10% Ficoll interface, whereas a smaller proportion sediments at the 10 to 15% Ficoll-400 interface. The total time required for vacuole isolation is 2 to 2.5 hours, beginning from leaf harvest.     

Analysis of polyamines in higher plants by high performance liquid chromatography

A sensitive (0.01-1 nmol) method has been developed for the analysis of polyamines in higher plant extracts based on high performance liquid chromatography (HPLC) of their benzoyl derivatives (Redmond, Tseng 1979 J Chromatogr 170: 479-481). Putrescine, cadaverine, agmatine, spermidine, spermine, and the less common polyamines nor-spermidine and homospermidine can be completely resolved by reverse phase HPLC, isocratic elution with methanol:water (64%, v/v) through a 5-μm C₁₈ column, and detection at 254 nm. The method can be directly applied to crude plant extracts, and it is not subject to interference by carbohydrates and phenolics. A good quantitative correlation was found between HPLC analysis of benzoylpolyamines and thin layer chromatography of their dansyl derivatives. With the HPLC method, polyamine titers have been reproducibly estimated for various organs of amaranth, Lemna, oat, pea, Pharbitis, and potato. The analyses correlate well with results of thin layer chromatography determinations.     

Separation and quantitation of phospholipids and their ether analogues by high-performance liquid chromatography.

The common mobile phase hexane/isopropanol/water used for separation of phospholipids on high-performance liquid chromatography silica columns poses several problems, such as incomplete separation and rapid column deterioration. By inclusion of 5 mM ammonium sulfate in the aqueous phase, we were able to substantially improve the chromatographic resolution and obtain complete separation of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidylglycerol, and sphingomyelin. In addition, ammonium sulfate prevented column degeneration and greatly improved reproducibility. A new quantitation method for alkenylacyl, alkylacyl, and diacyl forms of phospholipids was also developed based on derivatization with [(3)H]acetic anhydride. Separation and quantitation of the radioactive acetyl diradylglycerols were performed by straight-phase HPLC coupled to a radioactive flow detector and enabled detection of the various ether analogues at the picomole level with high reproducibility. The described methods are mild and nondestructive and can therefore be easily combined with analysis of either molecular species or fatty acid and aldehyde composition of the individual phospholipids.     

Quantitation and purification of polymerase chain reaction products by high-performance liquid chromatography

This work describes the application of the fully automated high-performance liquid chromatographic system to the analysis of PCR-amplified products. Efficient separations of both DNA restriction fragments and PCR products were performed using an anion-exchange DEAE-NPR column, packed with 2.5-μm nonporous particles. The automated HPLC method was employed for the separation, quantitation, and purification of PCR products in less than 10 min in a single step.     

The isolation of potato virus S from the leaves of potato plants using precipitation by polyethylene glycol

A purified potato virus S was prepared using precipitation by the solution of 35% polyethylene glycol 4000 in the presence of an electrolyte. The mixture for precipitation of the potato virus S had to contain 11% polyethylene glycol and 0·25m NaCl. The S virus was extracted by 0·01m phosphate buffer, pH 7·5, from the precipitation separated by centrifugation. One part of the extract was further purified by means of differential centrifugation and the other by means of gel filtration on Sephadex G-100. The ultraviolet absorption measurements of both preparations showed that the differential centrifugation gave a purer preparation than the gel filtration.