Secretory Mechanism of Fibroin,a Silk Protein,in the Posterior Silk Gland Cells of Bombyx mori

There are two microtubule-microfilament systems in the posterior silk gland cells of Bombyx mori. One is a radial microtubule system; the other is a circular microtubule-microfilament system. These two systems are presumably concerned with the intra-cellular transport of secretory granules of fibroin and the secretion of fibroin into the lumen, respectively. Conventional and scanning electron microscopic observations of the two microtubule-microfilament systems in the posterior silk gland cells are reported. Scanning electron micrographs showed that a number of parallel linear cytoplasmic processes ran circularly on the luminal surface of the posterior silk gland cells. These processes were assumed to correspond to the circular microtubule-microfilament systems. The effects of cytochalasin (B or D), a secretion stimulating agent of fibroin, on the intracellular recording of membrane potential from the posterior silk gland cells are also reported. Exposure to cytochalasin resulted in depolarization of the membrane potential of the gland cells. Possible functional roles of the two microtubule-microfilament systems in the secretory mechanism of fibroin are discussed with reference to the effects of antimitotic reagents and cytochalasin on these two systems.     

Promoter Characterization and Role of cAMP/PKA/CREB in the Basal Transcription of the Mouse ORMDL3 Gene

Orosomucoid 1-like 3 (ORMDL3) gene was strongly linked with the development of asthma in genetic association studies, and its expression could be significantly induced by allergen in airway epithelial cells of mice. However, the expression mechanism of ORMDL3 was still unclear. Here we have identified and characterized the mouse ORMDL3 gene promoter. Deletion constructs of the 5′ flanking region were fused to a luciferase reporter gene. After transient transfection in mouse fibroblast cell line NIH3T3, a CRE (−27/−20) binding CREB was identified in the core promoter region. Deletion or mutation of the CRE consensus sequence resulted in a significant loss of the promoter activity. EMSA and ChIP assays demonstrated the binding of CREB to the core promoter. Knocking down endogenous CREB led to a reduction in ORMDL3 expression. Conversely, overexpression of CREB up-regulated ORMDL3 expression. Moreover, forskolin, a PKA activator, could facilitate the phosphorylation of CREB, which in turn heightens ORMDL3 expression. H-89, a PKA-specific inhibitor, could significantly inhibit ORMDL3 expression. This study delineates the characterization of mouse ORMDL3 gene promoter and shows signaling pathway cAMP/PKA/CREB plays an important role in regulating ORMDL3 expression, which will be helpful for future animal model studies regarding the regulation or function of ORMDL3 gene.     

STUDIES ON THE POSTERIOR SILK GLAND OF THE SILKWORM, BOMBYX MORI : I. Growth of Posterior Silk Gland Cells and Biosynthesis of Fibroin During the Fifth Larval Instar

Growth of the posterior silk gland and biosynthesis of fibroin during the fifth larval instar of the silkworm, Bombyx mori, have been studied. In accordance with the exponential increase in the wet weight of the gland, the amounts of DNA, RNA, protein, and lipids per animal increased rapidly in the early stage of the fifth instar (0–96 hr). Biosynthesis of fibroin, on the contrary, mainly proceeds in the later stage of the fifth instar (120–192 hr). Electron microscopical observations have shown that, in the very early stage (0–12 hr), a number of free ribosomes exist in the cytoplasm. Rough endoplasmic reticulum (ER) with closely spaced cisternae was also observed. Then rough ER starts to proliferate rapidly, and at the same time lamellar ER is rapidly or gradually transformed into vesicular or tubular forms. In the later stage of the fifth instar (120–192 hr), the cytoplasm is mostly filled with tubular or vesicular ER. Golgi vacuoles, free vacuoles (fibroin globules), and mitochondria are also observed. It is concluded that in the early stage of the fifth instar the cellular structures necessary for the biosynthesis of fibroin are rapidly formed, while in the later stage the biosynthesis of fibroin proceeds at a maximum rate and utilizes these structures.     

Surface Modification and Characterisation of Silk Fibroin Fabric Produced by the Layer-by-Layer Self-Assembly of Multilayer Alginate/Regenerated Silk Fibroin

Silk-based medical products have a long history of use as a material for surgical sutures because of their desirable mechanical properties. However, silk fibroin fabric has been reported to be haemolytic when in direct contact with blood. The layer-by-layer self-assembly technique provides a method for surface modification to improve the biocompatibility of silk fibroin fabrics. Regenerated silk fibroin and alginate, which have excellent biocompatibility and low immunogenicity, are outstanding candidates for polyelectrolyte deposition. In this study, silk fabric was degummed and positively charged to create a silk fibroin fabric that could undergo self-assembly. The multilayer self-assembly of the silk fibroin fabric was achieved by alternating the polyelectrolyte deposition of a negatively charged alginate solution (pH = 8) and a positively charged regenerated silk fibroin solution (pH = 2). Finally, the negatively charged regenerated silk fibroin solution (pH = 8) was used to assemble the outermost layer of the fabric so that the surface would be negatively charged. A stable structural transition was induced using 75% ethanol. The thickness and morphology were characterised using atomic force microscopy. The properties of the self-assembled silk fibroin fabric, such as the bursting strength, thermal stability and flushing stability, indicated that the fabric was stable. In addition, the cytocompatibility and haemocompatibility of the self-assembled silk fibroin fabrics were evaluated. The results indicated that the biocompatibility of the self-assembled multilayers was acceptable and that it improved markedly. In particular, after the self-assembly, the fabric was able to prevent platelet adhesion. Furthermore, other non-haemolytic biomaterials can be created through self-assembly of more than 1.5 bilayers, and we propose that self-assembled silk fibroin fabric may be an attractive candidate for anticoagulation applications and for promoting endothelial cell adhesion for vascular prostheses.     

人SATB1基因5′上游序列的转录激活功能分析

在对人SATB1基因进行生物信息学分析的基础上 ,采用PCR技术 ,扩增人基因组DNA中SATB1基因 5′上游序列的 - 2 95 5～ - 9片段 ,构建了 3个分别由SATB1基因 5′上游 - 2 95 5～ - 9,- 172 7～ - 9和 - 76 0～ - 9序列片段驱动的报告载体 -pGL3 SP2 94 6 luc ,pGL3 SP1718 luc和pGL3 SP75 1 luc ,分别瞬时转染JurkatT ,K5 6 2 ,U937和HeLa细胞 ,通过测定荧光素酶的表达活性 ,观察SATB1基因 5′上游序列片段 3个删除突变体在不同细胞内活性的差异 .结果显示 ,SATB1上游序列- 2 95 5 - 9在 4种细胞中的转录激活能力为U937>JurkatT >K5 6 2 ,在HeLa细胞中基本无激活 ,提示SATB1的转录激活可能具有一定的细胞类型特异性 .3种 5′删除突变体转录激活性由大至小顺序为 - 76 0 - 9>- 2 95 5 - 9>- 172 7 - 9,提示SATB1的核心启动子可能存在于其 5′上游序列的- 76 0至 - 9bp区域中 .     

家蚕丝蛋白Fhx/P25基因启动子区域的克隆及序列分析

为研究家蚕Fhx/P25基因在时空上的调控机制,通过PCR扩增获得家蚕丝素蛋白Fhx/P25基因的启动子序列并进行克隆和序列分析。进一步构建了由Fhx/P25启动子驱动报告基因DsRed的表达载体pSK-P25-DsRed-PolyA,并通过家蚕BmN细胞进行瞬时表达。结果显示:Fhx/P25基因的启动子序列符合真核生物启动子特点,具有丝腺特异性表达启动子的特征,TATA框的保守序列为TATAA,位于-28—-32处,CAAT基序有3个,其中-110—-117和-90—-87处的2个CAAT基序可能具有活性;二级结构分析显示:Fhx/P25启动子区域具有复杂的茎环结构,这可能与蛋白表达的组织特异性、时间性以及活性有关。基因启动子可以驱动红色荧光基因DsRed在家蚕BmN培养细胞中的瞬时表达。     

组蛋白与转录因子在hAMFR基因启动子序列上的结合及相互作用

采用从鸡红细胞中分离纯化的组蛋白Ｈ１,核心组蛋白Ｈ２Ａ＋Ｈ２Ｂ和Ｈ３＋Ｈ４,以及从ＨｅＬａ细胞中萃取的含有ＲＮＡ聚合酶Ⅱ和多种Ⅱ类基因转录因子的可溶性ＨｅＬａ细胞核抽提物,通过凝胶迟滞电泳,对组蛋白和ＨｅＬａ细胞核抽提物中的转录因子在人自泌移动因子受体（Ｈｕｍａｎａｕｔｏｃｒｉｎｅｍｏｔｉｌｉｔｙｆａｃｔｏｒ,简称ｈＡＭＦＲ）基因上游启动子序列的相互作用关系进行了初步研究,得到以下结论,组蛋白Ｈ１     

Dynamic Rearrangement Within the Antheraea pernyi Silk Fibroin Gene Is Associated with Four Types of Repetitive Units

We characterized a full-length gene encoding wild silkmoth Antheraea pernyi fibroin (Ap-fibroin) to clarify the conformation of repetitive sequences. The gene consisted of a first exon encoding 14 amino acid residues, a short intron (120 bp), and a long second exon encoding 2,625 amino acid residues. Three amino acids, alanine, glycine, and serine, amounted to 81% of the Ap-fibroin sequence. The Ap-fibroin, except for 155 residues of the amino terminus, was composed of 80 tandemly arranged polyalanine-containing units (motifs). A motif was a doublet of a polyalanine block (PAB) and a nonpolyalanine block (NPAB). Seventy-eight of the 80 motifs were classified into four types based on differences in the NPAB sequences. Although respective motifs were significantly conserved, many rearrangements were observed within the second exon, i.e., the triplication of a 558-bp-long sequence and other duplication events of shorter sequences. Chi-like sequences, GCTGGAG, might contribute to the rearrangement within the gene as described in human minisatellite loci, because they were found at specific sites of NPAB-encoding sequences in three of four types of motifs. The present results support the idea that the Ap-fibroin gene is unstable like minisatellite sequences and that the evolution of this gene is strongly associated with its instability. Received: 18 February 2000 / Accepted: 30 June 2000     

组蛋白与hAMFR基因启动子的结合对体外转录活性的影响

采用从鸡血红细胞中分离纯化的组蛋白,从ＨｅＬａ细胞核中萃取的含有ＲＮＡ聚合酶Ⅱ和多种Ⅱ类基因转录因子的可溶性抽提物,以及从非洲爪蟾卵细胞核中撮以的萃取物热处理物上清用于核小体构建,以含有人自泌移动因子受体基因启动子序列的ＤＮＡ片段为模板进行体外转录实验,结果表明,组蛋白和转录因子在ｈＡＭＦＲ基因启动子序列上的竞争性结合对转录活性具有重要的影响作用,在启动子区域预先构建的核小体能够抑制转录活性;     

乙型肝炎病毒的核心启动子各区段功能的研究

将一系列核心启动子区的缺失突变引入乙肝病毒（ＨＢＶ）线性转录单元,从病毒的抗原,ＲＮＡ以及子代ＤＮＡ等各个水平,分析了各缺失突变对前基因组ＲＮＡ和前核心ＲＮＡ转录的影响,对核心启动子各片段的功能进行了深入的研究。Ｃ片段缺失后检测不到ｅ抗原和前核心ＲＮＡ,却仍有核心抗原和前基因组ＲＮＡ的合成以及病毒子代ＤＮＡ的复制;而Ｂ３片段缺失后ｅ抗原和核心抗原均有显著下降,但仍能检测到两种ｍＲＮＡ的合成和病毒子     

人SATB1基 5'上游序列的转录激活功能分析

在对人SATB1基因进行生物信息学分析的基础上,采用PCR技术,扩增人基因组DNA中SATB1基因5'上游序列的-2955～-9片段,构建了3个分别由SATB1基因5'上游-2955～-9,-1727～-9和-760～-9序列片段驱动的报告载体-pGL3-SP2946-luc,pGL3-SP1718-luc和pGL3-SP751-luc,分别瞬时转染Jurkat T,K562,U937和HeLa细胞,通过测定荧光素酶的表达活性,观察SATB1基因5'上游序列片段3个删除突变体在不同细胞内活性的差异.结果显示,SATB1上游序列-2955/-9在4种细胞中的转录激活能力为U937>Jurkat T>K562,在HeLa细胞中基本无激活,提示SATB1的转录激活可能具有一定的细胞类型特异性.3种5'删除突变体转录激活性由大至小顺序为-760/-9>-2955/-9>-1727/-9,提示SATB1的核心启动子可能存在于其5'上游序列的-760至-9 bp区域中.