甘三酯立体专一分析的研究：（Ⅱ）甘三酯立体专一分析方法初探

在第一部分综合讨论的基础上对甘三酯立体专一分析方法作了初步探讨。以液体油(菜油)及固体脂(猪脂)为基质,系统地研究了这一分析方法,取得了经验并补充了一些具体验证方法。分析液体油的结果与文献数据相一致,分析猪脂的结果欠佳,还存在一些问题需待进一步研究。     

Link between Lipid Second Messengers and Osmotic Stress in Plants

Plants are subject to different types of stress, which consequently affect their growth and development. They have developed mechanisms for recognizing and processing an extracellular signal. Second messengers are transient molecules that modulate the physiological responses in plant cells under stress conditions. In this sense, it has been shown in various plant models that membrane lipids are substrates for the generation of second lipid messengers such as phosphoinositide, phosphatidic acid, sphingolipids, and lysophospholipids. In recent years, research on lipid second messengers has been moving toward using genetic and molecular approaches to reveal the molecular setting in which these molecules act in response to osmotic stress. In this sense, these studies have established that second messengers can transiently recruit target proteins to the membrane and, therefore, affect protein conformation, activity, and gene expression. This review summarizes recent advances in responses related to the link between lipid second messengers and osmotic stress in plant cells.     

Hepatic patatin-like phospholipase domain-containing protein 3 sequence,single nucleotide polymorphism presence,protein confirmation,and responsiveness to energy balance in dairy cows

Patatin-like phospholipase domain-containing protein 3 (PNPLA3), commonly known as adiponutrin, is part of a novel subfamily of triglyceride lipase enzymes with potential effects on triglyceride metabolism in adipose and hepatic tissues. The predicted bovine PNPLA3 sequence has been identified, but expression of the gene had not been examined. The objectives of this study were to confirm the predicted bovine PNPLA3 gene sequence, determine expression of the bovine PNPLA3 gene in response to whole-animal energy balance, identify single nucleotide polymorphisms present in dairy cows, and verify the presence of the protein in the liver. Using liver biopsy samples collected from cows at +28 d relative to calving (DRTC), RNA was isolated and used to generate a cDNA template for amplification of the entire predicted coding sequence of PNPLA3 via PCR. To determine if energy balance alters the expression of PNPLA3, RNA was isolated and mRNA expression quantified in liver samples from mid-lactation cows after a 5-d ad libitum period (n = 5) and after a subsequent 5-d 50% feed restriction period (n = 5), and in samples collected from cows at −14, +1, +14, and +28 DRTC (n = 16). The presence of PNPLA3 protein was detected by Western blot in liver protein samples collected at +28 DRTC. Expression of hepatic PNPLA3 was decreased after a period of feed restriction (8.14 vs. 1.08 ± 2.17 arbitrary units, ad libitum vs. fasted). Expression of PNPLA3 mRNA was decreased at +1 and +14 DRTC compared with −14 DRTC (23.35, 7.28, 10.17, and 14.5 ± 4.9 arbitrary units, −14, +1, +14, and +28 DRTC, respectively). The presence of PNPLA3 protein was detected as a 55-kDa band in hepatic protein isolations from liver tissue collected at +28 DRTC. These data confirm the presence and sequence of the bovine hepatic PNPLA3 gene and single nucleotide polymorphisms. Furthermore, these data indicate responsiveness of bovine hepatic PNPLA3 to energy balance.     

磷脂酶在植物逆境胁迫信号传导中的作用

磷脂酰肌醇是构成细胞膜的重要组分,它在磷脂酶的作用下水解产生三磷酸肌醇、二酰基甘油、磷脂酸、溶血磷脂、不饱和脂肪酸等,不仅可以作为磷脂的合成原料,而且在细胞感受外界刺激及其信号传导过程中也起着重要作用.磷脂酰肌醇信号传导途径与光合作用、激素调控等影响植物生长发育过程及抗逆信号传导关系密切,由于磷脂酶的活性直接影响这些信号分子的产生,磷脂酶在磷脂酰肌醇信号传导途径中处于一个中心地位,根据磷酯不同水解位点所分类的磷脂酶PLA2,PLC和PLD具有多种同功酶形式,分别在植物生长发育的不同阶段及对外界环境因子应答的信号传导过程中起着重要的调节作用.     

磷脂酶A1水解蛋黄卵磷脂的研究

采用磷脂酶A1水解蛋黄卵磷脂,通过单因素实验和正交实验确定该酶水解蛋黄卵磷脂经济合理的工艺条件为：反应温度50℃,反应时间5h,加酶量0．40 IU／g,起始pH6．0,底物质量浓度80g／L;利用电喷雾飞行时间质谱仪分析水解前后蛋黄卵磷脂组分,磷脂酶A1水解Sn-1位脂肪酸,生成含Sn-2位不饱和脂肪酸的溶血磷脂,反应过程中有酰基位移发生。     

磷脂酶A2用于菜籽油脱胶

利用磷脂酶A2对菜籽油进行脱胶工艺研究,采用正交实验对磷脂酶脱胶的工艺条件进行了优化。结果表明,酶用量90μL/kg,pH6.0,含水量2%,反应温度40℃,反应时间3 h,最终含磷量小于10 mg/kg,达到了理想的脱胶效果。     

色褐链霉菌磷脂酶D基因pld的克隆与表达

利用表达载体pET-22b( ),实现了色褐链霉菌磷脂酶D基因在大肠杆菌BL21(DE3)中的高效表达.利用镍亲和柱对表达产物进行纯化,将纯化后的重组磷脂酶D作用于底物卵磷脂和丝氨酸定向合成磷脂酰丝氨酸,并用HPLC法检测酶活力.结果表明,目的蛋白可在短时间内进行大量表达.转酯反应6 h后卵磷脂的转化率达到31%,重组磷脂酶D活力达到39 U·(mg蛋白)-1.     

辅酶Q10经蛋白激酶A/胞浆型磷脂酶A2信号通路抑制血小板血栓素A2的生成

目的:血小板来源血栓素A2(thromboxane A2,TxA2)可诱发动脉粥样硬化和血栓形成,辅酶Q10(coenzyme Q10,CoQ10)可以抑制血小板活化、聚集和血栓形成.本实验旨在通过体外实验探讨CoQ10对血小板TxA2生成的影响及其调控机制.方法:用不同浓度(0、1、10、100?μmol/L)CoQ...     

Changes in phospholipase D experession in soybeans during seed development and germination

Activation of phospholipase D (PLD) has been linked to accumulation of nonhydratable phosphatides and lipid degradation leading to soybean seed deterioration during preharvest and postharvest events. This study examined the changes in PLD activity, protein, and mRNA in soybeans during seed development and germination. RNA blotting analysis indicated that expression of the gene that encodes PLD was highest during the early and middle stages of seed development. However, the amount of PLD activity accumulated per cotyledon reached the highest level in mature seeds. During germination and early seedling growth, PLD mRNA was not detected one day after imbibition, while a significant increase in PLD expression occurred in the cotyledons of three- and seven-day seedlings. Similarly, PLD activity and protein concentration showed little change during the first day of imbibition and increased afterward in three- and seven-day seedlings. These results suggested that expression of PLD is developmentally regulated and that the changes in its amount of activity and protein are controlled primarily at the mRNA level. Immunoblotting analysis further revealed the presence of PLD variants that were associated with specific stages of seed development and seedling growth. The PLD variants present in the cotyledons of mature seeds appeared to be distinct from those observed in the early stage of seed development and in young seedlings.     

Enzymatic Synthesis of 1‐Alkyl‐2‐hydroxy‐sn‐glycero‐2,3‐cyclic‐phosphate Using a Novel Lysoplasmalogen‐Specific Phopholipase D

Many phospholipase Ds (PLDs) are known to catalyze transphosphatidylation as well as hydrolysis of phospholipids. Transphosphatidylation of lysoplasmalogen (LyPls)‐specific phospholipase D (LyPls‐PLD), which catalyzes hydrolysis of ether lysophospholipids such as LyPls and 1‐hexadecyl‐2‐hydroxy‐sn‐glycero‐3‐phosphocholine (Lyso‐PAF), still remains unclear. This study aims to reveal the transphosphatidylation activity of LyPls‐PLD, that is, the production of cyclic ether lysophospholipid. The enzymatic reaction is conducted in a buffer system, and the reaction products of a novel LyPls‐PLD from Thermocrispum sp. are investigated using mass spectrometry (MS). MS analyses demonstrate the reaction products to consist of 100% 1‐hexadecyl‐2‐hydroxy‐sn‐glycero‐2,3‐cyclic‐phosphate (cLyPA) and choline from Lyso‐PAF; however, 1‐alkenyl‐2‐hydroxy‐sn‐glycero‐2,3‐cyclic‐phosphate from 1‐O‐1′‐(Z)‐octadecenyl‐2‐hydroxy‐sn‐glycero‐3‐phosphocholine and 1‐O‐1′‐(Z)‐octadecenyl‐2‐hydroxy‐sn‐glycero‐3‐phosphoethanolamine is not produced. These results are expected to help in elucidating the catalytic mechanism of LyPls‐PLD, that is, the rate‐limiting step, and indicate LyPls‐PLD to be useful for the one‐pot synthesis of cLyPA. Practical Applications: A novel phospholipase D, LyPls‐PLD, can exclusively synthesize cLyPA from Lyso‐PAF using a one‐step enzymatic reaction without an organic solvent. cLyPA could be expected to show bioactivities similar to those of cyclic phosphatidic acid, which promotes normal cell differentiation, hyaluronic acid synthesis, antiproliferative activity in fibroblasts, and inhibitory activity toward cancer cell invasion and metastasis.