Acyl Group Migrations in 2-Monoolein

Acyl migration in 2-monoolein dissolved in solvents under conditions common in lipid modification reactions has been studied. The effects on acyl migration of solvent, incubation temperature, water activity, polar additives and solid additives have been investigated. Extensive acyl migration occured in aliphatic hydrocarbons and water-miscible alcohols under dry conditions. The acyl migration rate could be decreased in several nonpolar solvents by adding a small amount of water or an alcohol. Increasing water activity had no effect in isooctane, but decreased the acyl migration rate dramatically in methyl tert-butyl ether and methyl isobutyl ketone. Several commonly used enzyme supports catalysed acyl migration, showing that supports with surface charges could catalyse acyl migration.     

PLD activation in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA

Pertussis toxin-insensitive GTP-binding protein was observed to be involved in prostaglandin F2α(PGF2α)-induced phosphoinositide metabolism in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA (CHO-PGF2α·R cells) (Ito, S. et al. Biochem. Biophys. Res. Commun. 200: 756, 1994). In the present study, we investigated PGF2α-induced PLD activation in CHO-PGF2α·R cells. PLD activation was examined by measuring the production of [³H]phosphatidylbutanol ([³H]PBut), a specific product of the PLD-catalyzed transphosphatidylation reaction. PGF2α-induced [³H]PBut formation was concentration-dependent with the maximal level obtained at 1 μM PGF2α. The maximal [³H]PBut formation was observed at 2 min after addition of PGF2α. Depletion of extracellular Ca²⁺ with EGTA suppressed PGF2α-induced PLD activation by 50%. PKC inhibitors Ro31–8425 and calphostin C inhibited PGF2α-induced [³H]PBut formation by 50%. PTK inhibitors genistein and herbimycin A failed to inhibit PGF2α-induced PLD activation. A combination of maximal effective concentrations of PGF2α (1 μM) and PMA (100 nM) enhanced PLD activation in an additive manner. Pretreatment of the cells with PMA for 2 h down-regulated PKCα and decreased PGF2α-induced PLD activation. These results suggest that PLD activation by PGF2α is mediated by both PKC-dependent and -independent pathways and that PKCα is involved in the former pathway.     

Characterization of rapeseed myrosinase-binding protein

Myrosinase-binding proteins (MBPs) were purified from seeds of Brassica napus L. (oilseed rape). The proteins were characterized with respect to amino-acid composition, peptide sequence and isoelectric points. Gel electrophoresis and Western blotting of protein extracts from mature seeds showed the existence of at least ten proteins reacting with a monoclonal anti-MBP antibody and ranging in molecular size from 110 to 30 kDa. Proteins other than MBP reacting with the anti-MBP antibody were assigned as myrosinase-binding protein-related proteins (MBPRPs). Two MBPRPs were purified by immunoaffinity chromatography and characterized with respect to partial amino-acid sequence. Sequence identities were found between MBP and MBPRP. Western blot analysis of protein extracts from different tissues of B. napus showed that MBPRP is present in the whole plant, whereas MBP mostly occurs in the mature seed. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to investigate the occurrence of MBP and MBPRP in developing seeds of some species in the Brassicaceae family.Abbreviations FPLC fast protein liquid chromatography - MBP myrosinase-binding protein - MBPRP myrosinase-bindingprotein-telated protein - PBS phosphate-buffered saline     

Structural and immunochemical identification of Leb glycolipids in the plasma of a group O Le(a-b-) secretor

Total non-acid glycosphingolipids were isolated from the plasma of a healthy red blood cell group O Le(a-b-) salivary ABH secretor individual. Glycolipids were fractionated by HPLC and combined into eight fractions based on chromatographic and immunoreactive properties. These glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with antibodies reactive to the type 1 precursor (Le^c), H type 1 (Le^d), Le^a and Le^b epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and LSIMS) and proton NMR spectroscopy. Expected blood group glycolipids, such as H type 1, (Fuc1-2Gal1-3GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified. Inconsistent with the red cell phenotype and for the first time, small quantities of Le^b blood group glycolipids (Fuc1-2Gal1-3(Fuc1-4)GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified in the plasma of a Lewis-negative individual. These findings confirm recent immunological evidence suggesting the production of small amounts of Lewis antigens by Lewis negative individuals. Abbreviations: HPLC, high performance liquid chromatography; TLC, (high performance) thin layer chromatography; EI-MS, electron impact ionisation mass spectrometry; LSIMS, liquid secondary ion mass spectrometry; NMR, nuclear magnetic resonance spectroscopy. The sugar types are abbreviated to Hex for hexose, HexNAc forN-acetylhexosamine and dHex for deoxyhexose (fucose). The ceramide types are abbreviated to d for dihydroxy and t for trihydroxy base, n for non-hydroxy and h for hydroxy fatty acids; LCB, long chain base.     

腺苷酸激酶基因在大肠杆菌中的可溶性高表达

报道了鸡肌腺苷酸激酶基因的克隆和在温控启动子P_RP_L调控下在大肠杆菌中的可溶性高效表达.SDS-PAGE分析表明,鸡肌腺苷酸激酶的含量可占大肠杆菌细胞总蛋白含量的38％.利用Johnson等的干冰/乙醇-冰水浴反复冻融法,可将此重组蛋白进行富集,纯度可达85％以上.鸡肌腺苷酸激酶可与抗兔肌腺苷酸激酶单克隆抗体产生强的交叉反应.     

在96孔板中进行抗脂质过氧化的微量测定

以Fe²⁺/半胱氨酸诱导大鼠肝微粒体为基本模型,根据硫代巴比妥酸(TBA)反应原理,优化不同反应条件,建立了一种在96孔板上进行抗脂质过氧化测定的一步反应方法,该方法的灵敏度不低于传统的试管法,而且还具有微量、快速、简便等优点,特别适用于大规模筛选和研究抗氧化剂.此外,也可用于其它系统诱导的抗脂质过氧化的测定.     

催产素在脊髓水平对电针镇痛的影响

采用玻璃微电极胞外记录和脊髓表面给药的方法观察了催产素（ＯＴ）、抗催产素血清（ＡＯＴＳ）以及电针穴位对背角神经元伤害性诱发放电的影响。结果表明：电针穴位或脊髓表面施加ＯＴ可部分抑制脊髓背角神经元的伤害性诱发放电;在电针的基础上施加ＯＴ则明显加强电针的抑制效应;相反,用ＡＯＴＳ预处理后,电针的抑制作用放取消。提示ＯＴ在脊髓水平参与了对痛觉信息的调制,并与一定频率的针刺镇痛有关。     

Cytotaxonomic study ofTragopogon L. in czechoslovakia

Karyotypes ofTragopogon orientalis L. subsp.orientalis, T orientalis L. subsp.leiocarpos (Sauter)Trnka,T. pratensis L.,T. minor Miller,T. dubius Scop. subsp.dubius andT. dubius Scop. subsp.major (Jacq.)Vollmann were studied. The occurrence in Slovakia ofT. pratensis was karyologically proved.     

The fluctuations in the level of endogenous growth regulators in seeds ofAcer pseudoplatanus L. in the course of stratification

In the course of cold stratification ofAcer pseudoplatanus L. fruits a statistically significant alternation occurs in their seeds of a rise and fall in the level of endogenous growth regulators. In the initial weeks the inhibitory effect slightly declines, or, on the contrary, the stimulatory effect slightly increases; in the middle phase of stratification a marked increase in inhibitions and reduction of stimulations appears, and towards the end of stratification the stimulatory effect of isolated substances in the individual biotests rises again, or their inhibition effect is decreased. No direct dependence was found between the decrease of the degree of dormancy and the drop of inhibitor, or increase of promotor levels. However, a certain analogy was observed between the time course of fluctuations in the level of growth regulators and the germination energy of the seeds investigated. An enhancement of the growth activity of the substances isolated (e.g. those of a gibberellin-like nature) in the last weeks of stratification can already be considered as the result of the release of fruits from dormancy.     

The influence of pH on the EPR and redox properties of cytochrome c oxidase in detergent solution and in phospholipid vesicles

The EPR signals of oxidized and partially reduced cytochrome oxidase have been studied at pH 6.4, 7.4, and 8.4. Isolated cytochrome oxidase in both non-ionic detergent solution and in phospholipid vesicles has been used in reductive titrations with ferrocytochrome c.The g values of the low- and high-field parts of the low-spin heme signal in oxidized cytochrome oxidase are shown to be pH dependent. In reductive titrations, low-spin heme signals at g 2.6 as well as rhombic and nearly axial high-spin heme signals are found at pH 8.4, while the only heme signals appearing at pH 6.4 are two nearly axial g 6 signals. This pH dependence is shifted in the vesicles.The g 2.6 signals formed in titrations with ferrocytochrome c at pH 8.4 correspond maximally to 0.25–0.35 heme per functional unit (aa₃) of cytochrome oxidase in detergent solution and to 0.22 heme in vesicle oxidase. The total amount of high-spin heme signals at g 6 found in partially reduced enzyme is 0.45–0.6 at pH 6.4 and 0.1–0.2 at pH 8.4. In titrations of cytochrome oxidase in detergent solution the g 1.45 and g 2 signals disappear with fewer equivalents of ferrocytochrome c added at pH 8.4 compared to pH 6.4.The results indicate that the environment of the hemes varies with the pH. One change is interpreted as cytochrome a₃ being converted from a high-spin to a low-spin form when the pH is increased. Possibly this transition is related to a change of a liganded H₂O to OH^? with a concomitant decrease of the redox potential. Oxidase in phosphatidylcholine vesicles is found to behave as if it experiences a pH, one unit lower than that of the medium.