Four-directional-development thin-layer chromatography of lipids using trimethyl borate

Solvent mixtures containing trimethyl borate virtually eliminated the pronounced interconversion of 1,2- and 1,3-dipalmitins during their resolution by thin-layer chromatography on Silica Gel G. With trimethyl borate, an average of 1-2% of 1,2-dipalmitin was converted to 1,3-dipalmitin. A four-directional-development TLC procedure incorporating trimethyl borate resolves cholesteryl glucoside, ceramides, monogalactosyl diglyceride, 1- and 2-monopalmitin, palmitic acid, cholesterol, 1,2- and 1,3-dipalmitin, tripalmitin, methyl palmitate, cholesteryl palmitate, beta-carotene and some of its degradation products, squalene, and tetracosane. Digalactosyl diglyceride, phosphatidic acid, phosphatidylglucose, cerebrosides, and other phospholipids remain near the origin. A mixture containing triolein, 1,2- and 1,3-diolein, 1- and 2- monoolein, oleic acid, and cholesterol was resolved in one dimension. A similar series of palmitic-containing neutral lipids was also resolvable in one dimension. These procedures were applied to the TLC of human sera lipids.     

银杏中种皮化学成分的分离及鉴定

银杏（Ginkgo biloba Linn．）在中国种植广泛,其产量占全世界总产量的70％[1]。中药白果仁是银杏种子去除肉质外种皮和骨质中种皮后得到的,具有敛肺定喘、止带缩尿的功效。目前,有关银杏种子的研究主要集中在具有药食同源的白果仁[2]及具有潜在应用价值的外种皮[3]上,鲜见对银杏种子中种皮的研究报道。生产中大量的银杏中种皮被废弃,因此,开展银杏中种皮的相关研究对于充分利用该类资源非常必要。鉴于此,作者对银杏中种皮的化学成分进行了系统分析,以期为其开发应用提供参考依据。     

蹄叶橐吾根的化学成分研究

从蹄叶橐吾（Ligularia fischeri）的根中分离得到13个化合物,通过波谱学等方法鉴定为2-hydrox-platyphyllide（1）,liguhodgsonal（2）,nsujapone（3）,6’-亚油酰基田-胡萝卜苷（4）,Luped（5）,6,7-二羟基香豆素（6）,3-谷甾醇（7）,胡萝卜苷（8）,（25,3S,4R,12E）-N-[2’-（R）-羟基二十二碳酰基]-1,3,4-三羟基-2-氨基-二十碳-12-烯（9）,单硬脂酸甘油酯（10）,α-羟基二十四烷酸（11）,1,3-二棕榈酸甘油酯（12）,二十七烷醇（13）。其中化合物1—6为首次从该种植物中分离得到。     

Synthesis of E- and Z-pyrazolylacrylonitriles and their evaluation as novel antioxidants.

A facile synthesis of (Z)- and (E)-2-(5-arylpyrazol-3-yl)-3-(pyrrol-2-yl)acrylonitriles and (Z)-2-(1,3-diarylpyrazol-5-yl)-3-pyrrol-2-yl)acrylonitriles, and isomerisation of (Z)-2-(5-arylpyrazolyl)acrylonitriles to (E)-2-(5-arylpyrazolyl)acrylonitriles under basic conditions have been reported. (Z)-2-(1,3-Diarylpyrazolyl)acrylonitriles did not undergo isomerisation under the similar conditions. New compounds were identified on the basis of their spectral data (1H-, 13C-, 1H-1H COSY, NOESY, NOE, HMQC NMR, IR, UV and EI mass). The structures of one acrylonitrile and five of their precursor 6-arylpyran-2-ones and cyanomnethylpyrazoles were confirmed by X-ray crystallographic studies. Effects of pyrazolylacrylonitriles and their precursors on rat liver-microsomal lipid peroxidation were evaluated in vitro with a view to establish structure activity relationship and to identify a lead compound.     

Subcellular distribution of steryl ester biosynthesis in spinach leaves

Higher steryl ester biosynthetic activities were obtained with Triton X-100-phosphatidylcholine-cholesterol mixed micelles than with Tween 80-phosphatidylcholine-cholesterol mixed micelles when incubated with spinach leaf (Spinacia oleracea L.) acetone powder preparations. The best incorporation of [4-¹⁴C]cholesterol into [4-¹⁴C]cholesteryl ester was obtained with a Triton X-100-phosphatidylcholine-cholesterol (10:1:1, w/w) mixed micelle system. This mixed micelle system, however, required 1,2-dipalmitin and fatty acid-free bovine albumin for optimal activity. The reaction exhibited a diglyceride specificity since the dipalmitin requirement could be replaced with neither 1-monopalmitin nor tripalmitin. Significant amounts of steryl ester biosynthetic activity were detected in the chloroplast (1,000g pellet), mitochondrial (3,000g pellet), and microsomal (20,000g and 88,000g pellet) fractions. Little activity was detected in the water-soluble (88,000g supernatant) fraction. The highest specific activity occurred in the 88,000g pellet. The 88,000g supernatant contained a heatstable, water-soluble substance that was required for optimal steryl ester biosynthesis in all of the pellet fractions. This factor was not lost during extensive dialysis but was destroyed by ashing, indicating that it was large and organic. Silver nitrate thin layer chromatography indicated that 60% of the biosynthesized steryl esters contained saturated fatty acids in the absence of 1,2-dipalmitin and that 83% contained saturated fatty acids in the presence of 1,2-dipalmitin.     

Rates and efficiencies of reactions of ruminal biohydrogenation of linoleic acid according to pH and polyunsaturated fatty acids concentrations

Data from a previous study about the effects of pH and of linolenic acid (C18:3n-3) and linoleic acid (C18:2n-6) concentrations on C18:2n-6 biohydrogenation in ruminal cultures were used to calculate the rates and efficiencies of the three reactions of C18:2n-6 biohydrogenation (isomerisation of C18:2n-6 to CLA; reduction of CLA to trans-octadecenoic acids; reduction of trans-octadecenoic acids to stearic acid). First, low pH was confirmed to inhibit isomerisation and was shown to inhibit the second reduction, leading to an accumulation of vaccenic acid. This later effect had only been observed in some in vivo studies using high concentrate diets, because in in vitro experiments, the very low pH frequently used depresses isomerisation which consequently generates very low amount of substrates for reductions whose variations become difficult to ascertain. Second, C18:2n-6 at high concentration was confirmed to saturate its own isomerisation and the increase of CLA production due to high initial C18:2n-6 was shown to inhibit the two subsequent reductions. Third, C18:3n-3 at high concentrations was confirmed to inhibit C18:2n-6 isomerisation. Moreover, the second reduction was shown to be saturated, probably by all trans-octadecenoic acids intermediates of C18:2n-6 and C18:3n-3 biohydrogenation, leading to an accumulation of trans-octadecenoic acids, especially vaccenic acid. This fatty acid is partly desaturated into CLA in the mammary gland, which explains the synergy between C18:2n-6 and C18:3n-3 for milk CLA noticed by others in vivo. This approach helped explain the actions of pH and of C18:2n-6 and C18:3n-3 concentrations on C18:2n-6 biohydrogenation and allows some explanations about differences noticed between studies.     

Phospholipid-nucleoside conjugates. 5. The interaction of selected 1-beta-D-arabinofuranosylcytosine-5'-diphosphate-L-1,2-diacylglycerols with serum lipoproteins

The phospholipid-nucleoside conjugates 1-beta-D-arabinofuranosylcytosine-5'-diphosphate-L-1,2-dipalmitin (1), -distearin (2), and -diolein (3) have been shown to interact rapidly with canine high density lipoprotein and with both high density and low density lipoproteins isolated from human serum. The extent of interaction with the high density lipoproteins appears to be dependent upon the characteristic gel-liquid crystalline phase transition of the conjugate's phospholipid. Since the phospholipid-nucleoside conjugates under study represent sustained release forms of the antileukemic agent 1-beta-D-arabinofuranosylcytosine, the therapeutic efficacy of these conjugates should now be considered in light of these interactions.     

Acyl specificity in triglyceride synthesis by lactating rat mammary gland.

We have studied the specificity of the acyl-CoA:diglyceride acyltransferase reaction in lactating rat mammary gland to provide a rational explanation at the enzyme level for the nonrandom distribution of fatty acids in milk fat triglycerides. Acyl-CoA:diglyceride acyltransferase activity was measured using various diglyceride and radioactive acyl-CoA substrates; products were identified as triglycerides by thin-layer and gas-liquid chromatography. Most of the enzymatic activity was located in the microsomal fraction and showed a broad specificity for the acyl donors tested C10, C12, C14, C16, C18, and C18:1 CoA esters). The acyltransferase activity was highly specific for sn-1,2-diglyceride enantiomers; rac-1,3- and sn-2,3-diglycerides were relatively inactive. The acyl-CoA specificity was not affected by the type of 1,2-diglyceride acceptor offered, although dilaurin was the best acceptor and sn-1,2-dilaurin greater than sn-1,2-dimyristin greater than sn-1,2-dipalmitin greater than sn-1,2-distearin. We have previously shown that in the microsomal fraction from lactating rat mammary gland, the acyltransferase activities concerned with the conversion of sn-glycero-3-phosphate to diacylglycerophosphate show a very marked specificity for long chain acyl-CoA's. Therefore, we conclude that the predominant localization of long chain fatty acids in the 1 and 2 positions, and of shorter chain fatty acids in the 3 position of the glycerol backbone, results at least in part from the specificities of the mammary gland acyltransferases.     

Evidence supporting a cis-enediol-based mechanism for Pyrococcus furiosus phosphoglucose isomerase

The enzymatic aldose ketose isomerisation of glucose and fructose sugars involves the transfer of a hydrogen between their C1 and C2 carbon atoms and, in principle, can proceed through either a direct hydride shift or via a cis-enediol intermediate. Pyrococcus furiosus phosphoglucose isomerase (PfPGI), an archaeal metalloenzyme, which catalyses the interconversion of glucose 6-phosphate and fructose 6-phosphate, has been suggested to operate via a hydride shift mechanism. In contrast, the structurally distinct PGIs of eukaryotic or bacterial origin are thought to catalyse isomerisation via a cis-enediol intermediate. We have shown by NMR that hydrogen exchange between substrate and solvent occurs during the reaction catalysed by PfPGI eliminating the possibility of a hydride-shift-based mechanism. In addition, kinetic measurements on this enzyme have shown that 5-phospho-d-arabinonohydroxamate, a stable analogue of the putative cis-enediol intermediate, is the most potent inhibitor of the enzyme yet discovered. Furthermore, determination and analysis of crystal structures of PfPGI with bound zinc and the substrate F6P, and with a number of competitive inhibitors, and EPR analysis of the coordination of the metal ion within PfPGI, have suggested that a cis-enediol intermediate-based mechanism is used by PfPGI with Glu97 acting as the catalytic base responsible for isomerisation.     

Effects of solvent and ionic medium on the kinetics of axial ligand substitution in vitamin B12. Part IV. The reaction between aquocobalamin and the thiocyanate ion in acetonitrile-water mixtures

The rate constants for the reaction of aquocobalamin with the thiocyanate ion were measured as a function of ionic strength and solvent composition in acetonitrile-water mixtures. The reaction is described by a two-step mechanism: the ligation reaction, where the most stable isomer (S-bonded) is formed and the isomerisation reaction (S-bonded to N-bonded thiocyanate). For the ligation reaction a full quantitative analysis of solvent effects could be performed, whereas for the isomerisation reaction only qualitative observations were made. The equilibrium constant for the isomerisation (S-bonded/N-bonded) is large and does not change with the solvent composition. It is found that the transfer Gibbs energies of activation for the ligation reaction are the same as found for the ligand thiourea. The absence of a solvent effect on the isomerisation reaction is a further example of the ability of vitamin B₁₂ to create its own micro environment.     

Non-equilibrium melting of amylose-V complexes

Thermal analysis of solution-grown crystalline amylose-V complexes of amylose, amylodextrin and β-cyclodextrin with a series of saturated 1-monoglycerides (C₁₀–C₁₈), lysolecithin, lauric acid and 1,3-dipalmitin was carried out using differential scanning calorimetry (DSC). At intermediate water contents (60%) and moderate heating rates (10°C min⁻¹), some of these complexes exhibited two melting transitions separated by an exothermic effect. A mechanism of partial melting, followed by recrystallization and final melting, is proposed to account for such non-equilibrium melting. These phenomena are strongly influenced by the amount of water present in the system; the higher the moisture content, the more cooperative the melting and the lower the occurrence of secondary crystallization processes. The overall thermal behavior of the complex/H₂O mixture can be explained in terms of both thermodynamic melting point depression due to the diluent and structural reorganization upon heating. The latter is related to the crystallite morphology/habit of the complex and appears to be dictated by the nature of the ligand molecule. Good amylose complexing agents induce metastable, less perfected crystalline structures that are inclined to reorganization upon heating in the DSC, presumably via a lamella thickening mechanism. Due to the non-equilibrium character of the melting process of these complexes, theoretical treatment of melting data using expressions such as the Flory-Huggins equation is not applicable.     

The formation and Langmuir-Blodgett deposition of monolayers of novel photochromic azobenzene-containing phospholipid molecules

A group of novel phospholipid molecules has been synthesised, in which one or both acyl chains has been replaced with an azobenzene-containing acid. These lipids photochromic, isomerising from the trans form in which the azobenzene unit is almost linear to a cis form with a bent chain. The influence of this isomerisation on the properties of monolayers of these lipids on an aqueous subphase has been investigated using the Langmuir trough. The lipids are shown to form compact monolayers when in the trans form, but to have greater molecular areas after isomerisation. All lipids could be transferred to a glass substrate by Langmuir-Blodgett deposition. Evidence suggestive of phase equilibria is seen for surface monolayers of one of the lipids, which also shows evidence of a temperature-dependent phase transition by light scattering when dispersed as vesicles in water. Marked hysteresis is seen between cycles of compression and expansion for surface monolayers of azo-lipids before and after photoisomerisation.     

Cis/trans isomerisation of unsaturated fatty acids in a cardiolipin synthase knock-out mutant of Pseudomonas putida P8

The gene encoding cardiolipin synthase ( cls) from the phenol-degrading bacterium Pseudomonas putida P8, which rapidly adapts its membrane lipids to the presence of organic solvents by cis/trans isomerisation of unsaturated fatty acids, was isolated and completely sequenced. The functionality of the predicted gene product was proven by constructing a knock-out mutant that was significantly reduced in its growth rate both at elevated temperatures and in the presence of membrane-active solvents. Though the mutant showed a clear phenotype it was still able to synthesise trace amounts of cardiolipin. As an increase in cardiolipin (diphosphatidylglycerol) content is known to function as a long term membrane adaptation mechanism in pseudomonads, we tested whether the mutant compensates for the lack of the Cls by increased cis/trans isomerisation of unsaturated fatty acids. Increase in cis/trans isomerisation of unsaturated fatty acids was observed for the mutant at zero and low concentrations of 4-chlorophenol; however, cis/trans isomerisation is not able to fully compensate for the lack of cardiolipin production. Possibly, other long-term adaptation mechanisms are instrumental in compensating for the missing cardiolipin synthesis. As the cis/trans isomerase is activated similarly in the mutant and the wildtype, cis/trans isomerisation and cardiolipin production do not display mutual dependency.     

The enzyme-catalyzed formation of bilirubin diglucuronide by a solubilized preparation from cat liver microsomes

When bilirubin monoglucoronide is incubated with a preparation from the 105 000 × g-supernatant of deoxycholate-treated cat liver microsomes, bilirubin diglucuronide is formed. This is an UDPglucuronate-dependent reaction whereby bilirubin IXα monoglucuronide is stoichiometrically converted into bilirubin IXα diglucuronide.The pH optimum for the conversion of bilirubin into bilirubin monoglucuronide lies between pH 8.0 and pH 8.8. For the conversion of mono- into diglucuronide two optima were found, one at about pH 6.5 and another at pH 8.1.When incubation was performed at pH 6.5 and the enzyme protein concentration was lowered, bilirubin monoglucuronide started to isomerise. As a result of this isomerisation bilirubin diglucuronide is also formed. Diglucuronide formation according to this mechanism however, can be clearly differentiated from the enzyme-catalyzed diglucuronide formation.By the formation of bilirubin monoglucuronide, one monoglucuronide isomer is preferentially synthesized.The alkaline-labile bilirubin conjugates in the bile of cats and rats have mainly the IXα isomeric structure. This suggests that in these animals bilirubin diglucuronide is formed enzymically as the bilirubin moiety of diglucuronide, formed by means of the isomerisation reaction, has predominantly the XIIα structure.     

反义RNA对猪α-1,3-半乳糖苷转移酶活性的影响

 α 1,3 半乳糖表位是猪 人异种移植超急性排斥反应的主要抗原 ,由α 1,3 半乳糖苷转移酶催化合成 .用RT PCR方法扩增中国实验用小型猪α 1,3 半乳糖苷转移酶cDNA的前 582bp ,测定碱基序列并构建其反义表达载体pLXRN ,将其转染入猪主动脉内皮细胞 .NorthernBlotting表明α 1,3 半乳糖苷转移酶mRNA减少 .检测α 1,3 半乳糖苷转移酶活性表明 ,反义RNA可使其活性下降32 2 % .研究结果表明可能通过反义RNA来抑制猪 人异种移植超急性排斥反应     

The synthesis,characterization, and preliminary biological evaluation of 1-β-D-arabinofuranosylcytosine-5′-diphosphate-L-1,2-dipalmitin

Recently, 1-β-$\text{D}$-arabinofuranosylcytosine-5′-diphosphate-$\text{DL}$-1,2-dipalmitin (VIa) was reported to inhibit the growth of L51784 cells in mice and of human colon carcinoma HCT-15 cells, also in mice. This paper describes the synthesis of a single diastereomer by conversion of 1-β-$\text{D}$-arabinofuranosylcytosine 5′-monophosphate (II) to the nucleoside 5′-phosphomorpholidate (III), followed by reaction with $\text{L}$-α-dipalmitoylphosphatidic acid (IV) to give 1-β-$\text{D}$-arabinofuranosylcytosine-5′-diphosphate-$\text{L}$-1,2-dipalmitin (V) in good yield. The separation of the product is described and its characterization by chromatography, elemental analysis, and spectroscopic methods. The lipophilic nature of V renders it insoluble in aqueous media and a method of sample preparation utilizing sonication techniques is described which provides a clear solution suitable for biological evaluation. In addition, the ability of V to inhibit the $\text{in}\text{vitro}$ growth of L1210 cells and of mouse myeloma MPC 11 cells is desscribed and compared with 1-β-$\text{D}$-arabinofuranosylcytosine (I) and other lipophilic prodrugs of I.     

Higher-order polarizabilities of polymethine systems in ground and excited electronic states

The electronic higher-order polarizabilities of linear and cyclic polymethine systems withelectron donor and acceptor groups included in the conjugation systems, in the ground and first excited singlet and triplet states, are studied using semiempirical quantum-chemical calculations (MNDO and PPP-DCI). It is shown that these polarizabilities are determined by two main factors: the bond order alternation in the conjugated system and the magnitude of the electron transfer within the molecule. The effect oftrans-cis isomerisation of the linear polymethines is also studied.     

Enzymatic synthesis of poly-N-acetyllactosamines as potential substrates for endo-beta-galactosidase-catalyzed hydrolytic and transglycosylation reactions

Enzymatic synthesis of GlcNAc-terminated poly-N-acetyllactosamine beta-glycosides GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(n)Galbeta1,4GlcNAcbeta-pNP (n=1-4) was demonstrated using a transglycosylation reaction of Escherichia freundii endo-beta-galactosidase. The enzyme catalyzed a transglycosylation reaction on GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (1), which served both as a donor and an acceptor, and converted 1 into p-nitrophenyl beta-glycosides GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(1)Galbeta1,4GlcNAcbeta-pNP (2), GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(2)Galbeta1,4GlcNAcbeta-pNP (3), GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(3)Galbeta1,4GlcNAcbeta-pNP (4) and GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(4)Galbeta1,4GlcNAcbeta-pNP (5). When 2 was used as an initial substrate, it led to the preferential synthesis of nonasaccharide beta-glycoside 4 to heptasaccharide beta-glycoside 3. This suggests that 4 is directly synthesized by transferring the tetrasaccharide unit GlcNAcbeta1,3Galbeta1,4GlcNAcbeta1,3Gal to nonreducing end GlcNAc residue of 2 itself. The efficiency of production of poly-N-acetyllactosamines by E. freundii endo-beta-galactosidase was significantly enhanced by the addition of BSA and by a low-temperature condition. Resulting 2 and 3 were shown to be useful for studying endo-beta-galactosidase-catalyzed hydrolytic and transglycosylation reactions.     

Decarboxylation of malonyl-CoA and biosynthesis of mevalonic acid in rat liver]

Biosynthesis of mevalonic acid (MVA), total formation of 14CO2 from [1,3-14C]malonyl-CoA and the activity of malonyl-CoA decarboxylase in subcellular fractions of rat liver were studied. The dependence of the rate of MVA biosynthesis on malonyl-CoA concentration was found to be linear both in 140,000 g supernatant and solubilized microsomal fractions. It was shown that in a composite system (140,000 g supernatant fraction added to washed microsomes, 10 : 1) the optimal concentration ratio for the substrates of MVA biosynthesis (malonyl-CoA and acetyl-CoA) is 1 to 2. In the absence of acetyl-CoA decarboxylation of [1,3-14C]malonyl-CoA was prevalent. In all subcellular fractions studied decarboxylation of [1,3-14C]malonyl-CoA prevailed over its incorporation into MVA, total non-saponified lipid fraction and fatty acids. The degree of malonyl-CoA, decarboxylation was not correlated with the rate of its incorporation into MVA, i. e. the increase in the 14CO2 formation was not accompanied by stimulation of [1,3-14C]malonyl-CoA incorporation either into MVA or into total non-saponified lipid fractions. The incorporation of [1-14C]acetyl-CoA into MVA under the same conditions was considerably lower than that of [1,3-14C]malonyl-CoA. In all subcellular fractions under study the activity of malonyl-CoA decarboxylase was found. The experimental data suggest that a remarkable part of malonyl-CoA is incorporated into MVA without preliminary decarboxylation. A possible role of malonyl-CoA decarboxylase as an enzyme which protects the cell against accumulation of malonyl-CoA and its immediate metabolites -- malonate and methylmalonyl-CoA is disucssed.     

Comparison of cytidinediphospho-sn-1,2-diglyceride transfer from microsomal and liposomal to mitochondrial membranes

Transfer of [3H]CDP-diglycerides from isolated guinea pig liver microsomal and liposomal membranes to guinea pig mitochondrial membranes was studied by incubating microsomal or liposomal membranes carrying [3H]CDP-diglycerides with mitochondrial membranes and determining the CDP-diglyceride-dependent incorporation of sn-3-[14C]glycerolphosphate into mitochondrial [14C]polyglycerophosphatides. A significant difference in the amount of transferred [3H]CDP-diglycerides and the composition of mitochondrial [14C]polyglycerophosphatides was found depending on whether [3H]CDP-diglycerides were transferred from microsomal or liposomal membranes. This amount was around 12% when [3H]CDP-diglycerides were transferred from the microsomal membranes and around 4.6% when they were transferred from the liposomal membranes. Furthermore, about 60% of [14C]phosphatidylglycerol and 35% of [14C]phosphatidylglycerophosphate were found in the microsomes-mitochondria system and about 9% of [14C]phosphatidylglycerol and 79% of [14C]phosphatidylglycerophosphate were found in the liposomes-mitochondria system, establishing an important role for the membrane donor in the transfer of [3H]CDP-diglycerides to mitochondria. Furthermore, if the transfer of [3H]CDP-diglycerides from the microsomal to the mitochondrial membranes was assayed by the determination of [3H]CDP-diglycerides in reisolated mitochondrial membranes without further incorporation into mitochondrial polyglycerophosphates, it amounted to about 38%.