大肠杆菌胞嘧啶脱氨酶基因的克隆及其真核表达载体的构建

目的 克隆大肠杆菌胞嘧啶脱氨酶(CD)基因，构建真核表达载体，本研究拟探索该基因在肿瘤基因治疗中的应用基础。方法 根据GenBank数据库提供的CD基因核苷酸序列，设计并合成一对引物，采用PCR方法，从大肠杆菌基因组DNA中扩增出CD基因，并与pcDNA3．1定向连接，构建受控于人巨细胞病毒启动子的重组真核载体pcDNA3．1-CD，并用限制性内切酶、PCR和DNA测序进行鉴定。结果克隆了大肠杆菌CD基因，并构建了真核表达载体，经限制性内切酶酶切、PCR扩增和DNA测序证实了其正确性。结论 pcDNA3．1-CD真核表达载体构建成功。     

考虑热量和压力功影响的氢网络扩展矩阵法

以解藻酸弧菌株（Vibrio alginolyticus）ATCC 17749为研究对象,利用生物信息学寻找并预测得到5个可能的海藻酸裂解酶基因algV1、 algV2、 algV3、 algV4和 algV5。通过构建5个以pET 28a(+)为载体的大肠杆菌表达质粒pET28a algV1、pET28a algV2、pET28a algV3、pET28a algV4和pET28a algV5,实现了5个基因的异源表达,并经海藻酸裂解酶定量和定性的活性分析,确定5个基因的编码产物都具有海藻酸裂解酶活性,其中重组的algV1、 algV2和 algV3为胞外酶,algV4和algV5为胞内酶。     

The 3-O-sulfation of heparan sulfate modulates protein binding and lyase degradation

Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.

Heparan sulfates (HSs) are highly sulfated polysaccharides that reside on the surface and in the extracellular matrix of virtually all cells of multicellular organisms (1, 2). A large number of proteins, including blood coagulation factors, growth factors and morphogens, chemokines and cytokines, proteins involved in complement activation, and cell adhesion and signaling proteins can bind to HS, resulting in conformational changes, stabilization of receptor−ligand complexes, protein oligomerization, sequestration, and protection against degradation (3, 4). These molecular recognition events regulate many physiological processes, including embryogenesis, angiogenesis, blood coagulation, and inflammation. These interactions are also important for many disease processes such a cancer, viral and bacterial infections, neurological disorders, and a number of genetic diseases (1–4).The biosynthesis of HS starts with the assembly of a protein-bound polymer composed of alternating N-acetyl glucosamine (GlcNAc) and glucuronic acid (GlcA) residues. Discrete regions of this polymer are modified by N-deacetylase/N-sulfotransferases to replace N-acetyl by N-sulfate moieties. Subsequently, the regions of N-sulfation are further modified by a C-5 epimerase that converts GlcA into iduronic acid (IdoA), followed by O-sulfation by iduronosyl 2-O-sulfotransferase (2-OST), glucosaminyl 6-O-sulfotransferases (6-OST), and 3-O-sulfotransferases (3-OST) (5).HS modifications are often incomplete, resulting in at least 20 different HS disaccharide moieties, which can be combined in different manners, creating considerable structural diversity (4, 6). The way these disaccharides are arranged is not random but dictated by the substrate specificities of the HS biosynthetic enzymes. These enzymes are present in multiple isoforms, each having unique substrate specificity. It has been hypothesized that, by regulating the expression of the isoforms of HS biosynthetic enzymes, cells can create unique HS epitopes (4, 7). The so-called “HS sulfate code hypothesis” is based on the notion that such epitopes can recruit specific HS-binding proteins, thereby mediating multiple biological and disease processes.Vertebrates express seven 3-OST isozymes in a cell- and tissue-selective manner. It is the largest family of HS-modifying enzymes, indicating that, in concert with other sulfotransferases, they have an ability to create unique epitopes for recruitment of specific proteins (8). A prototypic example of a protein requiring binding to 3-O-sulfated HS is antithrombin-III (AT-III) to confer anticoagulant activity. The pentasaccharide GlcNAc6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S has been identified as high-affinity ligand for AT-III (9). Removal of the sulfate at C-3 of N-sulfoglucosamine (GlcNS3S) results in a 10⁵-fold reduction in binding affinity. The context in which the 3-O-sulfate (3-OS) is presented is also important, and removal of any other sulfate leads to a substantial reduction in binding affinity. Glycoprotein D (gD) of HSV-1 is another example of an early discovered protein that binds to HS epitopes having a 3-O-sulfated GlcNS residue (10). The interaction of gD with HS in concert with other viral envelope proteins is critical for triggering fusion with the host cell surface membrane. Although HS oligosaccharides have been used to probe ligand requirements of gD, the optimal carbohydrate sequence remains to be determined. A number of other proteins are known to require a 3-O-sulfated HS epitope for binding and biological activity, and examples include neuropilin-1 (Nrp-1) (11), cyclophilin B (12), stabilin (13), receptor for advanced glycosylation end product (RAGE) (8), and fibroblast growth factor-7 (FGF-7) (14). The context in which the 3-OS group needs to be presented for optimal binding is largely unknown. It is the expectation that many other proteins need a 3-O-sulfoglucosamine moiety for binding and biological activity. In this respect, this modification has been implicated in many physiological and disease processes, including cell differentiation, axon guidance and growth of neurons, inflammation, vascular diseases, and tumor progression, yet HS-binding proteins that are involved in these diseases are often not known (8).There are indications that 3-O-sulfation can interfere in the degradation of HS by heparin lyases (15, 16). These enzymes are critical for the analysis of heparin/HS (Hep/HS) by controlled depolymerization into smaller fragments, which can then be more readily analyzed by various methods including mass spectrometry (MS) (17). Furthermore, a mixture of lyases I, II, and III can digest heparin into disaccharides facilitating compositional analysis (18). Lyases are also employed for the production of low molecular weight heparins (LMWHs) with higher anticoagulant activities and improved pharmacokinetic profiles (19). Treatment of heparin with a mixture of lyases I, II, and III results in the formation of “resistant” trisaccharides and tetrasaccharides that usually contain a 3-OS moiety (15). These studies have been performed with heparin, which is structurally less diverse compared to HS, and, as a result, there is limited knowledge of how a 3-O-sulfation impacts the degradation of HS by lyases (19).The rudimentary understanding of ligand requirements of HS-binding proteins requiring a GlcNS3S moiety is, in part, due to the fact that this modification is relatively rare and difficult to detect and analyze. This is compounded by a lack of robust technologies that can establish the importance of a 3-OS moiety for binding and biological activity. It is the expectation that a sufficiently large collection of synthetic HS oligosaccharides with and without a 3-OS will provide a powerful discovery tool to establish ligand requirements of 3-OS−binding proteins. Such a collection of compounds will also be valuable to define substrate specificities of heparin lyases and other HS-processing enzymes.Although synthetic approaches to prepare HS oligosaccharides have progressed (7, 20), the preparation of a sufficiently large collection of compounds is still challenging, and careful consideration should be given to compounds selection. A literature survey indicates that GlcNS3S moieties can be flanked by different types of uronic acids, and typical sequences include GlcA-GlcNS3S-GlcA (13, 21), IdoA-GlcNS3S-GlcA (22), IdoA2S-GlcNS3S-GlcA (23–25), GlcA-GlcNS3S-IdoA (13, 22, 23), IdoA-GlcNS3S-IdoA (22), IdoA2S-GlcNS3S-IdoA (25), GlcA-GlcNS3S-IdoA2S (13, 23, 26–28), and IdoA2S-GlcNS3S-IdoA2S (24, 29, 30) (Fig. 1A). In these sequences, the GlcNS3S moieties can be further modified by a sulfate ester at C-6. Such a modification is preferentially installed by specific isozymes, and, for example, it is known that 3-OST-1 prefers substrate having 6-OS on GlcNS, whereas 3-OST-3 has a higher activity for compounds with a hydroxyl at this position (24). To corroborate the importance of 3-OS for binding, it is also important to prepare structural counterparts without such a functionality.Open in a separate windowFig. 1.(A) Identified substructures having 3-OS−bearing glucosamine. (B) General structure of target HS hexasaccharides, featuring structurally diverse sequence (shaded pyranose rings), site of sulfation (text in red), uronic acid composition (wavy bond at C-5 carboxylic acid), constant reducing end GlcN and nonreducing end disaccharide, and an anomeric linker for fabrication of HS arrays. (C) Hexasaccharides numbering and backbone composition, variable core trisaccharide in red color; NS, N-sulfate; 2S: 2-OS; 3S, 3-OS; 6S, 6-OS. (D) Disaccharide building blocks comprising acceptors 10 to 12 and donors 13 to 19 for modular assembly of hexasaccharides.Based on these considerations, 27 synthetic HS oligosaccharides were designed based on nine different core trisaccharides encompassing all relevant uronic acid modifications (Fig. 1 B and C). The central GlcNAc moiety of the nine templates is modified by either a 3-OS, 6-OS, or 3,6-OS and extended at the reducing and nonreducing end by GlcNS6S and GlcA-GlcNS6S, respectively to give sufficiently large set of compounds for binding and enzymology studies. It is expected that, after hit identification, the constant regions can be further optimized in a systematic manner to identify the optimal ligand.     

一个三组妹同患P450c17α缺陷家系的分子遗传学研究

研究１个三组妹同患Ｐ４５０ｃ１７α缺陷家系的分子遗传学机制。方法采用聚合酶链反应－单链构象多态性，限制性内切酶酶切及自动测序等方法检测患者家系中ＣＹＰ１７基因的突变情况。结论１个三组妹同时患病的中国人Ｐ４５０ｃ１７α缺陷的家系是由于ＣＹＰ１７基因的复合杂合突变所致。     

胞嘧啶脱氨酶基因对移植静脉平滑肌增生的抑制作用

目的　研究胞嘧啶脱氨酶基因 / 5 -氟胞嘧啶自杀基因系统对移植静脉平滑肌增生的抑制作用 .方法　将含 Ad-CMVCD(CMV为启动子、含胞嘧啶脱氨酶基因的腺病毒载体 )的病毒上清加压注入兔颈外静脉管腔 (两端及分支结扎 ) ,半小时后剪下该静脉并用 Cuff技术移植于兔颈动脉上 ,以单纯移植组和 Ad CMVL acz(含 β-半乳糖苷酶基因的重组腺病毒载体 )为对照 ,4 wk后取静脉桥行 RT- PCR以确定转染效率 .通过光镜和电镜观察平滑肌细胞的形态变化 ,并对血管桥外径、管腔、内膜、中膜面积和外弹力膜周长及管腔相对丧失率进行定量分析 ,统计学处理以观察抑制内膜增生、防治血管再狭窄的效果 .结果　治疗组的内膜平滑肌增生明显 <两个对照组 .管腔丧失率亦明显减少 ,Ad CMVCD组为 (3.6 8±0 .4 2 ) % ,单纯移植组为 (9.6 8± 0 .4 1) % ,(P<0 .0 1) .结论　Ad CMVCD自杀基因系统对移植静脉平滑肌的增生有抑制作用 .     

大鼠脑、肝和前列腺一氧化氮合酶mRNA表达

根据大鼠脑型一氧化氮合酶（ｂＮＯＳ）和内皮型一氧化氮合酶（ｅＮＯＳ）的ｃＤＮＡ序列分别设计特异引物，用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）方法检测２种ＮＯＳ在脑、肝脏和前列腺中的ｍＲＮＡ表达差异。结果表明：ｂＮＯＳ在小脑、大脑皮质和海马中有表达，并且在肝脏和前列腺组织中也有表达：ｅＮＯＳ在肝组织中有表达，在小脑、大脑皮质和海马等脑组织中均有表达，但前列腺组织中未检测到ｍＲＮＡ表达。２种ＮＯＳ在上述组织中的广泛分布提示ＮＯ具有多种复杂的、有时甚至是相反的生理功能可能与ｂＮＯＳ和ｅＮＯＳ甚至诱导型一氧化氮合酶（ｉＮＯＳ）分别或共同作用有关。