肌肉脂肪酸转运膜蛋白及其相互关系

肌肉（骨骼肌）组织对脂肪酸的利用水平是影响机体能量稳态的关键因素.肌肉摄取的长链脂肪酸(long chain fatty acids,LCFAs)主要依赖细胞膜载体蛋白协助的跨膜转运过程.近年来,一系列与脂肪酸转运相关的膜蛋白被相继克隆鉴定,其中在肌肉中大量表达的有脂肪酸转运蛋白-1（fatty acid transport protein-1,FATP-1）、膜脂肪酸结合蛋白（plasma membrane fatty acid binding protein,FABPpm）、脂肪酸转位酶（fatty acid translocase,FAT/CD36）和小窝蛋白-1（caveolin-1）.研究上述肌肉脂肪酸转运膜蛋白的结构功能、调控机制及相互关系,可能为肥胖等脂类代谢紊乱疾病的诊治提供新的手段.     

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。     

肝型脂肪酸结合蛋白研究进展

肝型脂肪酸结合蛋白（liver fatty acid binding protein,L-FABP）是脂肪酸结合蛋白（fatty acid binding proteins,FABPs）家族重要的成员,在肝脏、小肠、肾脏等组织中均有表达。L-FABP在不饱和脂肪酸、饱和脂肪酸、胆固醇、胆汁酸等转运过程中扮演重要角色。目前研究显示L-FABP在脂肪肝、肝硬化以及肝癌发生发展中起到重要作用,并有望作为肝损伤的早期检测指标。此外,新近研究发现尿中L-FABP水平还可以用于预测1型糖尿病患者的临床结局。在2型糖尿病中,尿中L-FABP与糖尿病性肾病的病程有密切关系。主要就L-FABP的特性、结构及其与疾病的关系做一综述。     

齐口裂腹鱼和鲤鱼H-FABP基因的克隆及其表达谱

心型脂肪酸结合蛋白（heart fatty acid binding protein, H-FABP）的水平与影响肉质性状的肌内脂肪含量有关,鱼类H-FABP的表达水平对其肌内脂肪含量是否相关仍未见报道.本研究获得齐口裂腹鱼和鲤鱼心脏型脂肪酸结合蛋白基因序列,利用半定量RT-PCR分析其表达特性并测定肌内脂肪含量,比较H-FABP基因在不同生活环境的2种鲤科鱼肌内脂肪沉积中的作用.结果显示,齐口裂腹鱼和鲤鱼H-FABP基因的ORF为402 bp,编码133个氨基酸,它们的氨基酸序列相同,与人、猪、小鼠、斑马鱼、大西洋鲑、虹鳟等的同源性为71.3%~ 90%;H-FABP基因在2种鲤科鱼的心、肌肉、脂肪、肝、脑、脾、肾和鳃等组织中均有表达,肝中的表达量显著高于其它组织（P<0.05）,H-FABP基因的肌肉表达谱在齐口裂腹鱼和鲤鱼中存在明显差异：齐口裂腹鱼中的表达随生长发育呈上升趋势,在大体重鱼（500 g）中的表达显著高于小体重鱼（P<0.05),其表达与肌内脂肪含量呈显著正相关（R=0.370,P<0.05）;H-FABP基因在鲤鱼生长发育中呈下降趋势,而小体重鱼（50~60 g）中的表达显著高于其它大体重鱼(P<0.05),其表达与肌内脂肪含量呈显著负相关（R=-7.083,P<0.01）.据此推测,齐口裂腹鱼和鲤鱼肌肉组织H-FABP基因表达与肌内脂肪关联性的差异可能与2种鱼的生活环境不同有关.     

巴西橡胶树(Hevea brasiliensis)微管相关蛋白全长cDNA克隆及表达分析

从巴西橡胶树Hevea brasiliensis差减cDNA文库中分离到微管相关蛋白（Microtubule-associated protein,MAPs）基因片段,根据该基因片段序列信息,设计特异引物,采用cDNA末端快速扩增技术RACE（Rapid Amplification ofcDNA Ends）进行差异片段的5＇和3＇端的扩增,获得了长度为788bp的全长cDNA,该基因在GenBank中的登录号为AY461412.序列分析表明该基因包含完整的开放阅读框,编码144个氨基酸,与微管相关蛋白基因家族具有很高的同源性,推测该基因是微管相关蛋白基因.半定量RT-PCR检测证实它在胶乳中的表达强于叶中,胁迫处理（伤害及乙烯处理）使其表达上调.     

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。     

光皮桦ACC氧化酶基因BlACO的克隆和表达分析

ACC（1-aminocyclopropane-1-carboxylic acid）氧化酶（ACO）是植物乙烯合成过程中的关键限速酶,对乙烯的合成具有重要的调控作用。以光皮桦茎叶组织提取的RNA为模板,据已报道的ACO同源序列设计简并引物,通过RT-PCR扩增获得部分基因片段,结合5＇,3＇RACE方法从光皮桦中扩增出1个ACO的全长cDNA序列。该基因cDNA全长1262bp,具有一个957bp的完整开放阅读框架,编码含318个氨基酸的蛋白。与其他植物中的ACO基因进行同源性比对的结果显示,BlACO蛋白与欧洲白桦的同源性最高,达到97%。该基因在光皮桦的雄花和雌花中表达量较高,而在茎中的表达量较低。     

甘蔗乙烯合成酶基因家族三个成员的克隆与序列分析

ACC（1-aminocyclopropane-1-carboxylic acid）合成酶是高等植物乙烯生物合成途径中的限速酶.根据已克隆的植物ACS（1-aminocyclopropane-1-carboxylic acid synthase）基因同源序列,设计简并引物,以甘蔗叶片总DNA为模板,通过PCR扩增,得到3条特异性强的扩增片段：Sc-ACS1为1 041 bp、Sc-ACS2为1 345 bp和Sc-ACS3为1 707 bp.将序列在GenBank核酸数据库进行同源性搜索,结果表明,3个片段均为ACS基因,推导编码的蛋白质序列分别包含326、242和310个氨基酸.其中,Sc-A CS1和Sc-ACS3同源性最高,核苷酸序列和蛋白质氨基酸序列分别有98%和96%同源,与禾本科植物玉米Zm ACS6、水稻OS-ACS2、毛竹等ACS基因家族也有很高的同源性,核苷酸序列同源性为88%-98%,蛋白质氨基酸序列同源性为73%-81%.甘蔗Sc-ACS2与水稻OS-ACS5在核苷酸和氨基酸序列上分别有91%和79%同源性,但与甘蔗Sc-ACS1和Sc-ACS3基因成员之间,氨基酸同源性分别只有45%和49%.系统进化分析表明,Sc-ACS1和Sc-ACS3基因与玉米Zm ACS6基因亲缘关系最近,而Sc-ACS2基因与水稻OS-ACS5基因亲缘关系最近.Southern杂交表明三基因在基因组中确实存在而且是多拷贝基因.三个片段已在GenBank数据库中注册,注册号分别为AY620985、AY620986和AY788919.     

肝型脂肪酸结合蛋白研究进展

肝型脂肪酸结合蛋白(liver-type fatty acid binding protein,L-FABP,FABPI)是脂肪酸结合蛋白家族的成员之一,主要在肝脏、小肠、肾脏及胰腺等组织细胞中有表达.研究发现,L-FABP与脂肪酸的摄取、转运、代谢调节有关.近年研究表明,肝型脂肪酸结合蛋白(L-FABP)与肿瘤、肾脏疾病、脂肪肝、肥胖、糖尿病等多种疾病的发生发展密切相关.本文就肝型脂肪酸结合蛋白的分子结构、功能以及与疾病的关系作一综述.     

猪白细胞介素-18基因克隆、序列分析及其真核表达载体的构建

采集6月龄河南良杂猪的脾脏,分离淋巴细胞后直接提取总RNA,进行反转录-聚合酶链反应（RT-PCR）扩增,扩增产物进行T-A克隆、测序,获得了河南良杂猪IL-18全基因序列,序列测定表明,plL-18全基因核苷酸长度为579bp,编码 192个氨基酸。与GenBank上已发表序列ABO10003进行比较,核苷酸同源性为99.8%,在第550位处 (以ATG为1计)由A→G,存在有意义突变。与GenBank下载读取的ABO10003、AF176949、AY262109、NM1997序列进行比较分析,氨基酸同源性分别为99%、98.5%和99.8%、99%。将该基因片段克隆到真核表达载体pcDNA3.1,构建重组质粒pcDNA-ILl8m,所获重组质粒经过酶切、测序鉴定,证实含有目的片段,且连接、构建正确,为核酸疫苗的研究应用奠定了基础。     

Cloning and characterization of chicken adipocyte fatty acid binding protein gene

Fatty acid binding proteins (FABPs) are members of a superfamily of lipid-binding proteins and occur intracellularly in vertebrates and invertebrates. This study was designed to clone and characterize the adipocyte fatty acid binding protein (A-FABP) gene in the chicken. PCR primers were designed according to mammalian A-FABP gene sequence to amplify partial cDNA of A-FABP gene from chicken adipose tissues, and the full length of the gene was cloned by 5'RACE and 3'RACE. Analysis of sequence showed that the cDNA of the chicken A-FABP gene was 74 and 73% homologous with porcine and human A-FABP gene, respectively. The similarity was 77, 28, and 23% at the predicted amino acid level with human A-FABP, human L-FABP, and human I-FABP, respectively. RT-PCR and Northern blot analysis indicated that the chicken A-FABP gene, similar to that of the mammal, is only expressed in fat tissues. This is the first report to identify and characterize A-FABP gene in the chicken.     

Characterization of the porcine FABP5 gene and its association with the FAT1 QTL in an Iberian by Landrace cross

We have characterized and mapped the porcine fatty acid binding protein 5, epidermal (FABP5) gene. According to linkage and RH mapping, this gene is located close to the FABP4 (fatty acid binding protein 4, adipocyte) gene on swine chromosome 4. We resequenced 4.7 kb of the FABP5 gene in the parental population of an Iberian x Landrace cross (IBMAP), identifying seven SNPs arranged in two distinct FABP5 haplotypes. QTL and association analyses in the IBMAP population showed that this gene is strongly associated with fat deposition. QTL and haplotype analysis revealed that both FABP4 and FABP5 (clustered in mammals) are major candidate genes for the FAT1 QTL; the most likely position for the FAT1 QTL is between these two genes. Finally, our results suggest the presence of more than one QTL affecting fatness traits on porcine chromosome 4.     

Amino acid sequence and some ligand binding properties of fatty acid-binding protein from bovine brain

Summary A fatty acid-binding protein (FABP) from the cytosol of bovine brain was purified by Sephadex G-75 filtration and electrofocusing. The purified protein migrated as a single protein band in 15% polyacrylamide gel electrophoresis with an apparent molecular mass of 14.7 kDa. To ascertain that the purified protein was a FABP, it was submitted to fatty acid-binding tests. Oleic and palmitic acids bound to brain FABP but this was not the case for palmitoyl CoA. By Scatchard analysis the ligand binding values were: Kd = 0.28 µM, Bmax (mol/mol) = 0.6 for oleic acid and Kd = 0.8 µM, Bmax (mol/mol) = 2.1 for palmitic acid. The complete amino acid sequence of the brain FABP was determined and a microheterogeneity was observed. Sequence comparison with other FABPs of known sequence and the observed microheterogeneity demonstrated the presence in brain of several homologous FABPs closely related to heart FABP.This paper corresponds to a communication at the first international workshop on fatty acid binding proteins (Maastricht, the Netherlands, September 4–5, 1989).     

FABP4基因在阿勒泰羊尾脂沉积与代谢模型中的表达变化规律

FABP4(Fatty acid binding protein 4)参与细胞内脂肪酸的转运和脂肪酸代谢,是当前研究动物脂肪沉积与代谢的热门候选基因。为了研究FABP4基因在绵羊尾脂沉积与代谢中的作用,文章采用生物信息学方法分析了FABP4氨基酸序列在各物种中的保守性;利用半定量RT-PCR方法检测了该基因在阿勒泰羊主要组织中的表达;采用饥饿法成功建立了模拟阿勒泰羊尾脂沉积与代谢的动物模型,利用qPCR和iTRAQ(isobaric tags for relative and absolute quantitation)技术同时验证FABP4基因mRNA和蛋白在阿勒泰羊非饥饿组与饥饿组尾脂中的表达变化。序列分析结果表明,绵羊FABP4氨基酸序列在物种间高度保守,提示FABP4基因可能由于其重要的生物功能而在进化中表现出其物种的保守性。组织表达谱结果显示,FABP4 mRNA在阿勒泰羊肠脂与尾脂中均高丰度表达,暗示FABP4基因可能在脂肪中行驶着重要的生理生化功能。qPCR与iTRAQ结果显示,FABP4 mRNA与蛋白在两种极端条件下尾脂中的表达差异均不显著(P>0.05),表明FABP4基因可能不是绵羊尾脂沉积与代谢两种极端差异表型的决定基因。以上研究结果为进一步研究FABP4基因在绵羊尾脂中的生物功能奠定了基础。     

Distribution of fatty acid binding proteins in tissues and plasma of Gallus domesticus

1. Fatty acid binding activity associated with a 14,000-15,000 mol. wt protein was observed in the cytosolic fraction of liver, duodenum, myocardium, adipose, pectoral and gastrocnemius muscles of chickens. 2. Polyclonal antisera prepared against chicken liver fatty acid binding protein affinity for only liver FABP and a 14,000 mol. wt fatty acid binding protein in the intestine. 3. A fatty acid binding protein was not detected in chicken plasma.     

猪L-FABP基因的克隆、表达特征及遗传多态性研究

FABPs属于脂结合蛋白超家族成员,是一类分子量较小而对脂肪酸有高亲和力的蛋白质,广泛存在于脊椎动物和非脊椎动物的细胞质中.FABPs担当细胞内脂肪酸的运输任务,它们与脂肪酸结合将其运输到脂肪酸氧化的位置、脂肪酸脂化成甘油三醋或磷脂的位置,或者进入细胞核内发挥其可能的调控功能.因此FABPs对脂类代谢具有重要的调控作用.本研究把L-FABP基因作为影响猪肌内脂肪含量的候选基因.为此,利用cDNA末端快速扩增(RACE)和PCR技术,克隆到猪肝脏型脂肪酸结合蛋白基因(L-FABP)的全长cDNA序列(GenBank登录号AY960623)和部分基因组序列(GenBank登录号DQ182323).猪L-FABP基因的cDNA序列全长518 bp,该序列包括起始密码子TGA和38 bp的5'末端非编码区(5'URT),终止密码子TAG和99 bp的3'末端非编码区(3'URT),在3'URT结构区域中包含polyA加尾信号序列AATAAA.猪L-FABP基因与其他FABPs基因一样,也由4个外显子(67 bp、173 bp、93 bp和51 bp)和3个内含子组成,内含子1和3的大小是1 679bp和565 bp,没有获得内含子2的序列,外显子和内含子剪接处符合GT/AG规律.应用Clustal W/X程序对猪L-FABP与其他物种的L-FABP进行多重序列比对,发现猪L-FABP与人、大鼠、鸡的L-FABP的相似性分别为89.8%、81.9%和72.4%.亲水性分析表明,猪L-FABP也是一个潜在的跨膜蛋白,在氨基酸残基57-65之间有一个明显的跨膜α螺旋.应用半定量RT-PCR分析发现,猪L-FABP在猪体组织中广泛存在,但在肝脏和小肠组织中表达量最为丰富.分析还发现,所克隆得到的编码区核苷酸序列与已知猪L-FABP基因的编码区核苷酸序列存在一定的变异,分别是外显子2中T→C(116位)、C→T(231位)、C→A(236位)和A→C(258位),演绎成氨基酸在Leu74Met存在差异.为进一步证实这些突变位点在猪群中真实存在,利用PCR-SSCP检测方法对4个猪种(藏猪、大河猪、雅南猪和约克夏)的157头个体的外显子2全序列进行SNP位点多态性片段的基因型分型,结果发现一个C→T的单核苷酸多态,等位基因频率在中国地方猪种(藏猪、大河猪、雅南猪)与国外约克夏猪种间存在极显著的差异(P＜0.01).连锁分析发现,基因型CC的肌内脂肪含量(4.86±0.22%)显著的高于基因型CT(4.16±0.23%)和TT(4.05±0.27%)的肌内脂肪含量(P＜0.05).因此,推测L-FABP基因可能是影响猪肌内脂肪含量的主效基因或与主效基因紧密连锁的标记基因,并且能够在分子标记辅助选择中用于对猪肌内脂肪含量的遗传改良.     

Primary structure of locust flight muscle fatty acid binding protein.

The amino acid sequence of the fatty acid binding protein (FABP) from flight muscle of the locust, Schistocerca gregaria, has been determined. The sequence of the N-terminal 39 amino acid residues, determined by automated Edman degradation, was used to prepare a degenerate oligonucleotide that corresponded to amino acid residues 16-23. cDNA coding for FABP was constructed from flight muscle mRNA and amplified by the polymerase chain reaction using the degenerate oligonucleotide and an oligo dT-NotI primer adapter as primers. The amplification product was cloned and sequenced. Additionally, a cDNA library of flight muscle mRNA was prepared and screened with a 414-bp probe prepared from the clone. The primary structure of locust FABP was compared with the proteins in the Swiss protein databank and found to have significant homology with mammalian FABPs over the entire 133-residue sequence. The best match was versus human heart FABP (41% identity), attesting to the highly conserved nature of this protein. The results suggest that locust muscle FABP is a member of the lipid binding protein superfamily and may provide valuable insight into the evolution of this abundant protein class.     

The complete amino acid sequence of porcine gastrotropin, an ileal protein which stimulates gastric acid and pepsinogen secretion

The stomach is stimulated by an enterooxyntin factor in a delayed response to feeding, resulting in an increase in both gastric acid and pepsinogen secretion. We have previously reported on the identity of such a factor from the porcine ileum (Wider, M. D., Vinik, A. I., and Heldsinger, A. (1984) Endocrinology 115, 1484-1491). This protein, termed gastrotropin, is localized to the distal region of the ileum where it constitutes less than 0.1% of the cytosolic protein. We have completed the primary structure of porcine gastrotropin by Edman degradation and mass spectrometry. Gastrotropin (Mr = 14,054) contains 127 amino acid residues and has a blocked (acetylated) alanine at its NH2 terminus. The sequence of porcine gastrotropin is similar to rat liver fatty acid-binding protein (FABP), with 44 of 127 residues being identical (35%). Homology with other members of the FABP family is significantly less apparent, with the order of similarity being liver FABP greater than heart FABP greater than retinol-binding protein greater than intestine FABP. The sequences of the NH2-terminal regions of these proteins account for virtually all of the homology; there are 9 conserved residues common to all five proteins. Gastrotropin represents the first member of the FABP family which has an extracellular function.