脂肪细胞型脂肪酸结合蛋白的研究进展

脂肪细胞型脂肪酸结合蛋白（adipocyte fatty acid binding protein,AFABP/aP2）作为脂肪酸结合蛋白（FABPS）超家族成员之一,广泛存在于各种正常的组织细胞中,参与脂肪酸贮存,运输与降解等过程。近年来,对脂肪细胞型脂肪酸结合蛋白的研究已成为热点,本文就其主要特征及其与各类疾病的关系作一简要综述。     

In VitroRearing ofTrichogramma minutumRiley (Hymenoptera: Trichogrammatidae) for Ten Generations, with Quality Assessment Comparisons ofin Vitroandin VivoReared Adults

Trichogramma minutumRiley were reared for 10 generations on an artificial diet containing a yeast extract, FreeAmine III, nonfat dry milk, chicken egg yolk, chicken embryo extract, andManduca sexta(L.) egg liquid. Quality control parameters, including adult longevity, sex ratio, pupation rate, percentage of pupae to emerge as adults, adult female body length, number ofHelicoverpa zea(Boddie) eggs parasitized by a female, and percentage of deformed females were assessed and compared to insects rearedin vivoon irradiatedH. zeaeggs. The development time was longer forin vitroreared insects, but there were more deformed females in thein vitroculture. The sex ratio, however, was generally not significantly different between thein vitroandin vivocultures. Thein vitroreared females generally were larger, lived longer, and parasitized moreH. zeaeggs. Emergence of adults was in excess of 75% in all but the firstin vitrogeneration and was generally not significantly different from adult emergence in thein vivoculture. These findings will be of value in the development of a practical system forin vitromass rearing ofTrichogrammafor use in biological control.     

Formation of Postsynaptic-Like Membranes during Differentiation of Embryonic Stem Cellsin Vitro

To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, α₁subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the γ- and ?-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR ?-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiationin vitroneuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by “loss of function” analysisin vitro.     

Syntheses and reactions of methyl (Z)-9,10-epoxy-13-oxo-(E)-11-octadecenoate and methyl (E)-9,10-epoxy-13-oxo-(E)-11-octadecenoate

The syntheses and reactions of two epoxyketoacids (methyl (Z)-9,10-epoxy-13-oxo-(E)-11-octadecenoate (IV) and methyl (E)-9,10-epoxy-13-oxo-(E)-11-octadecenoate (V)) are described. The synthetic method is based on the stereoselective oxidation of linoleic acid by soybean lipoxygenase to produce the corresponding 13-hydroperoxide. Reduction of the hydroperoxide with sodium borohydride followed by oxidation, esterification and epoxidation yielded the compounds IV and V with a global yield of 14% and 3%, respectively, referred to the diasteromerically pure isolated compounds. Confirmation of the structures was carried out by reduction of the ketone group with sodium borohydride and by the opening of the oxirane ring with methanolic boron trifluoride. The reduction of compounds IV and V with hydrogen mainly yielded the tetrahydrofuranoid fatty acid, methyl 10,13-epoxyoctadecanoate. This reaction may be considered a new procedure to obtain tetrahydrofuranoid fatty acids.     

Occurrence of 13(S)-Hydroxyoctadecadienoic Acid in Biological Samples

The oxygenated metabolite of linoleic acid, 13(S)-hydroxyoctadecadienoic acid has recently been shown to play a role in cellular regulation. To detect this molecule in biological systems, we recently developed a specific polyclonal antibody. Using this antibody, we report the presence of 13(S)-hydroxyoctadecadienoic acid in human urine, cell culture media, and untreated goat serum for the first time by a specific, sensitive, and rapid enzyme immunoassay. Furthermore, the enzyme linked immunosorbent assay data are verified by gas chromatography/mass spectrometry analysis of the same samples.     

Extracellular fatty acid binding protein (Ex-FABP) modulation by inflammatory agents: "physiological" acute phase response in endochondral bone formation

Ex-FABP, extracellular fatty acid binding protein, is a 21 kDa lipocalin expressed in hypertrophic cartilage, muscle and heart during chick embryo development and in granulocytes. Ex-FABP synthesis was increased in chondrocyte and myoblast cultures by inflammatory agents (LPS; IL6) and repressed by antiinflammatory agents. Expression of Ex-FABP and specific gelatinases is paralleled in hypertrophic cartilage; LPS specifically induced high molecular weight gelatinase ( > 200 kDa). LPS-treated hypertrophic chondrocytes showed increased chemotactic activity for endothelial cells paralleled by increased expression of transferrin. A high amount of Ex-FABP was expressed in adult pathological cartilage both in dyschondroplastic and osteoarthritic chickens. Controls were negative. Ex-FABP could represent a stress protein physiologically expressed in tissues where active remodelling is taking place during development and in tissues characterized by an acute phase response due to pathological conditions. We also suggest that during endochondral bone formation other responses characteristic of a local inflammatory status, such as gelatinase production and angiogenic factor secretion, are "physiologically" activated.     

脂肪酸合成酶在肿瘤中表达的调节机制

脂肪酸合成酶（fatty acid synthase,FASN）是肿瘤脂质生成的一种关键酶,在催化脂肪酸合成的最后一步中发挥重要作用。FASN在许多肿瘤细胞中过表达而在相应的正常细胞中却不表达。有证据表明FASN是一个代谢性癌基因,在癌细胞中高表达,在肿瘤生长和存活中有重要的作用。FASN在肿瘤中的表达调节是一个很复杂的过程,包括转录水平、翻译后控制和微环境状态的影响。正确认识FASN在肿瘤中的表达调节机制和研究新的FASN抑制剂,为成功治疗肿瘤提供一种新的思路。     

猪I-FABP基因的分子克隆与组织特异性表达分析

小肠型脂肪酸结合蛋白对长链脂肪酸具有高度的亲和力,参与脂肪酸的吸收和细胞内转运。利用cDNA末端快速扩增（RACE）技术并结合同源克隆策略,克隆到了编码猪小肠型脂肪酸结合蛋白基因（I-FABP）的全长cDNA序列（GenBank接受号：AY960624）,并对系统发育关系等进行了生物信息学分析。猪I-FABP基因的cDNA序列全长614 bp,其中包括399bp的开放式读码框（ORF）,43bp的5’末端非编码区（5’URT）和172bp的3’末端非编码区（3’URT）,编码132个氨基酸残基蛋白,在氨基酸水半上与其他物种的I-FABP具有高度的同源性。以邻接法（Neigbor-Joining,NJ）所构建的系统发育关系表明,猪I-FABP与其他物种的,I-FABP属于同一类群,且与人的遗传距离最近。Northern杂交和半定量RT—PCR分析发现,猪I-FABP在猪体组织中出现约620bp大小的转录本,且在猪体组织中广泛存在,但在小肠组织中表达量最为丰富。     

Probing the Interaction of Brain Fatty Acid Binding Protein (B-FABP) with Model Membranes

Brain fatty acid-binding protein (B-FABP) interacts with biological membranes and delivers polyunsaturated fatty acids (FAs) via a collisional mechanism. The binding of FAs in the protein and the interaction with membranes involve a motif called “portal region”, formed by two small α-helices, A1 and A2, connected by a loop. We used a combination of site-directed mutagenesis and electron spin resonance to probe the changes in the protein and in the membrane model induced by their interaction. Spin labeled B-FABP mutants and lipidic spin probes incorporated into a membrane model confirmed that B-FABP interacts with micelles through the portal region and led to structural changes in the protein as well in the micelles. These changes were greater in the presence of LPG when compared to the LPC models. ESR spectra of B-FABP labeled mutants showed the presence of two groups of residues that responded to the presence of micelles in opposite ways. In the presence of lysophospholipids, group I of residues, whose side chains point outwards from the contact region between the helices, had their mobility decreased in an environment of lower polarity when compared to the same residues in solution. The second group, composed by residues with side chains situated at the interface between the α-helices, experienced an increase in mobility in the presence of the model membranes. These modifications in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues (group II) that is likely to facilitate the delivery of FAs to target membranes. On the other hand, residues in group I and micelle components have their mobilities decreased probably as a result of the formation of a collisional complex. Our results bring new insights for the understanding of the gating and delivery mechanisms of FABPs.     

脂肪细胞型脂肪酸结合蛋白疫苗对高脂喂养雌鼠体重和糖耐量的影响

目的:构建能诱导出针对脂肪细胞型脂肪酸结合蛋白(FABP4)特异性中和抗体的疫苗,为高脂诱导下肥胖和胰岛素抵抗的防治新途径提供理论和实验依据。方法:野生型C57BL/6J雌鼠随机分为疫苗组(n=10,高脂饲养)、佐剂组(n=10,高脂饲养)和空白对照组(n=10,普通饲养),分别予以皮下注射生物合成的FABP4蛋白、佐剂和磷酸盐缓冲液,观察比较各组抗体滴度、安全耐受性和体重、摄食量、空腹血糖、胰岛素抵抗指数(HOMA-IR)、糖耐量实验血糖曲线下面积(AUC)等指标。结果:疫苗组小鼠产生了高滴度的FABP4特异性抗体,并于第3轮加强免疫后达到平衡状态。首次免疫16周后,疫苗组小鼠体重高于空白对照组,但明显低于佐剂组(P<0.05);日平均摄食量高于空白对照组(P<0.05),与佐剂组无差异(P>0.05);空腹血糖、HOMA-IR、腹腔葡萄糖耐量实验AUC均明显低于佐剂组(P<0.05),与对照组无统计学差异(P>0.05)。结论:以FABP4作为抗原免疫小鼠,可产生高滴度特异性抗体IgG,有效降低高脂喂养野生型雌性小鼠体重、空腹血糖、HOMA-IR和血糖AUC等指标,为高脂诱导的肥胖和胰岛素抵抗的治疗提供了新的途径和初步证据,可进行深入研究。     

脂肪酸脱氢酶基因在兔体内表达的生物学效应研究

目的研究脂肪酸脱氢酶基因在兔子体内的生物效应,探索不饱和脂肪酸与兔子免疫的相互作用。方法用壳聚糖(CS)和壳聚糖的聚乙烯亚胺(PEI)、聚乙二醇(PEG)修饰产物(mPEG-O-CS-PEI),对含有脂肪酸脱氢酶基因的真核表达质粒VRMFat-1进行包裹,肌肉注射新西兰白兔,每两周采取抗凝血,检测血液中免疫细胞数量的变化情况,利用气相色谱仪检测动物组织中脂肪酸的含量变化,并通过荧光定量PCR检测免疫细胞内免疫相关基因的表达情况。结果两组实验兔组织中的n-3不饱和脂肪酸含量显著高于对照组(P<0.05),其TLR4、CD3、IL-4和IL-6基因表达水平显著升高(P<0.05)。结论转入的脂肪酸去饱和脱氢酶基因能够在实验兔体内正常表达,初步证明不饱和脂肪酸对兔子的固有免疫和获得性免疫机能均有明显的增强作用。     

Free Fatty Acid Composition of Human and Rat Peripheral Nerve

Abstract: The free fatty acid (FFA) composition of peripheral nerve resembles that of erythrocytes but the composition of both is different from that of brain and other tissues. Approximately 75% of FFAs of nerve and erythrocytes are saturated and <5% are polyunsaturated whereas in brain and other tissues, 30-45% of FFAs are saturated and 25-50% are polyunsaturated. Approximately 10-15% of the total FFA of nerve have very long chain lengths [C₂₄, C₂₆, C₂₈, and C₃₀]. The presence of these very long-chain FFAs in endoneurium cannot be accounted for by the retention of erythrocytes or by lipid degradation. During Wallerian degeneration a significant increase of 18:1, associated with a decrease of saturated FFAs, was found in rat sciatic endoneurium, but normal values were approached when fiber regeneration was well under way. The FFA composition with chain length ≥C₂₆ were not, however, significantly altered with degeneration or repair of nerves. The metabolic significance of this striking difference between nerve and brain FFA composition is unknown but may reflect different functional properties.     

番茄内质网ω-3脂肪酸去饱和酶基因原核表达载体的构建与鉴定

通过RT-PCR的方法从番茄叶片克隆到内质网ω-3脂肪酸去饱和酶(LeFAD3)基因的部分编码区,该片段cDNA为309 bp,将其克隆到pET-30a(+)载体中,酶切位点分别是BamH I和Sac I,构建了原核表达载体pET-LeFAD3,并在大肠杆菌BL21中表达融合蛋白,经IPTG诱导蛋白表达,提取蛋白并采用SDS-PAGE和蛋白质免疫印迹法检测目的蛋白的表达情况.酶切鉴定结果表明,LeFAD3基因原核表达载体构建成功,目的蛋白成功表达.     

Fatty acid analyses of phosphoglycerides from tissues of the clam Chlamys islandica (Muller) and the starfish Ctenodiscus crispatus (Retzius) from Balsfjorden,Northern Norway

Fatty acid analyses of phosphoglycerides from Chlamys islandica (Muller) and Ctenodiscus crispants (Retzius) show several interesting features. Major phospholipids from the ovaries and muscles of Chlamys have high levels of (n-3) polyunsaturated fatty acids (PUFA). The phosphatidylinositol (PtdIns) from these tissues contains, however, large amounts of arachidonic acid, 20:4(n?6), so that the $\text{(n?3)}\text{(n?6)}$ ratio of PtdIns is considerably lower than that of the other phospholipids. In contrast, the major phospholipids from the ovaries of Ctenodiscus contain approximately equal amounts of (n?6), mostly 20:4(n?6), and (n?3)PUFA. The 20:4(n?6) is mostly in phosphatidylethanolamine (PtdEtn) and PtdIns and the $\text{(n?3)}\text{(n?6)}$ ratio is again lowest in PtdIns. The PtdIns in both species is also rich in 18:0 and consequently resembles PtdIns from elasmobranchs, marine teleosts, and terrestrial mammals. The results are discussed in relation to dietary origins of PUFA in benthic marine animals and to metabolic functions of PtdIns.