怀槐细胞悬浮培养的结构化动力学模型

为探明怀槐细胞生长、异黄酮染料木素合成与底物消耗间的关系,建立了怀槐细胞悬浮培养的结构化动力学模型。模型预测分析了胞内外的蔗糖代谢、胞内结构组分变化、胞内中间组分的变化、细胞呼吸损失以及胞内外异黄酮染料木素的合成情况。模型各参数灵敏度的分析表明kM_b1、k_b2 和k_p是最为灵敏的参数,其调节10%时,目标函数变化的最大比例分别达12.8%、4.61%和2.54%,其它参数对目标函数变化的影响均小于0.5%。该模型预测值与实验值具有较好的吻合性。     

Molecular cloning and analysis of a cDNA coding for the bifunctional dihydrofolate reductase-thymidylate synthase of Daucus carota

Molecular cloning of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota was achieved by immunoscreening of a cDNA library obtaining a 2 kbp clone which contains an open reading frame of 1528 bp. Comparison of the deduced amino acid sequence with those from other sources revealed the presence of motifs typical of DHFR and TS thus confirming the bifunctional nature of the carrot protein. As in other organisms, a higher degree of conservation was observed in the TS domain. Analysis of the dhfr-ts gene content in carrot revealed the presence of several copies per diploid genome.     

Spatiotemporal expression patterns of sialoglycoconjugates during nephron morphogenesis and their regional and cell type-specific distribution in adult rat kidney

The expression of 2,6- and 2,3-linked sialic acids on N-glycans was studied in embryonic, postnatal, and adult rat kidney. Histochemistry and blotting using Polyporus squamosus and Sambucus nigra lectins for 2,6-linked sialic acids and the Maackia amurensis lectin for 2,3-linked sialic acids were performed and sialyltransferase activity was assayed. N-glycans with 2,6- and 2,3-linked sialic acid were differently expressed in the two embryonic anlagen and early stages of nephron. Metanephrogenic mesenchyme was positive for 2,3-linked sialic acid but not for the 2,6-linked one, which became detectable initially in the proximal part of S-shaped bodies. Collecting ducts were positive for 2,6-linked sialic acid, whereas 2,3-linked sialic acid was restricted to their ampullae. Although positive in embryonic kidney, S1 and S2 of proximal tubules became unreactive for 2,3-linked sialic acid in postnatal and adult kidneys. In adult kidney, intercalated but not principal cells of collecting ducts were reactive for 2,3-linked sialic acid. In contrast, 2,6-linked sialic acids were detected in all cells of adult kidney nephron. Blot analysis revealed a different but steady pattern of bands reactive for 2,6- and 2,3-linked sialic acid in embryonic, postnatal, and adult kidney. Activity of 2,6 and 2,3 sialyltransferases was highest in embryonic kidney and decreased over postnatal to adult kidney with the activity of 2,6 sialyltransferase always being three to fourfold that of 2,3 sialyltransferase. Thus, 2,6- and 2,3-linked sialic acids are differently expressed in embryonic anlagen and mesenchyme-derived early stages of nephron and show regional and cell type-specific differences in adult kidney.     

鲢鱼轻酶解肌球蛋白的cDNA克隆及结构解析

与栖息温度相关的鲢鱼两种轻酶解肌球蛋白重链(light meromyosin,LMM)同工型(低温型,sc-w;高温型,sc-s)的氨基酸序列解析结果表明:sc-w与sc-s在LMM的氨基酸序列上显示91.8%的同源性,但与已经报道的草鱼(Ctenopharyngodon idella)低温型(gc10)有96.9%的...     

Plant Growth Retarding Activity of the 4-Substituted-7-(β-d-ribofuranosyl)pyrrolo[2,3-d]pyrimidine Anticytokinins

Three protease inhibitors (OTI-1-3) have been purified from onion (Allium cepa L.) bulbs. Molecular masses of these inhibitors were found to be 7,370.2, 7,472.2, and 7,642.6 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Based on amino acid composition and N-terminal sequence, OTI-1 and -2 are the N-terminal truncated proteins of OTI-3. All the inhibitors are stable to heat and extreme pH. OTI-3 inhibited trypsin, chymotrypsin, and plasmin with dissociation constants of 1.3×10^-9 M, 2.3×10^-7 M, and 3.1×10^-7 M, respectively. The complete amino acid sequence of OTI-3 showed a significant homology to Bowman-Birk family inhibitors, and the first reactive site (P₁) was found to be Arg¹⁷ by limited proteolysis by trypsin. The second reactive site (P₁) was estimated to be Leu⁴⁶, that may inhibit chymotrypsin. OTI-3 lacks an S-S bond near the second reactive site, resulting in a low affinity for the enzyme. The sequence of OTI-3 was also ascertained by the nucleotide sequence of a cDNA clone encoding a 101-residue precursor of the onion inhibitor.     

Isoforms of soybean seed oil body membrane protein 24 kDa oleosin are encoded by closely related cDNAs

We have characterized two cDNA clones for 24 kDa soybean oleosin, the seed oil body membrane protein. Differences in the predicted amino acid sequences of the two clones and the presence of a doublet on immunoblots indicate that 24 kDa oleosin exists in at least two isoforms in soybean. The predicted amino acid sequence also contains a unique carboxy terminal region that is dominated by a series of different tandem amino acid repeats.     

Isolation, characterization and molecular cloning of the bark lectins from Maackia amurensis

A detailed study was made of the bark lectins of the legume tree Maackia amurensis using a combination of protein purification and cDNA cloning. The lectins, which are the most abundant bark proteins, are a complex mixture of isoforms composed of two types of subunits of 32 and 37 kDa, respectively. Isolation and characterization of the homotetrameric isoforms indicated that the 32 kDa subunit exhibits a 100-fold stronger haemagglutinating activity than the 37 kDa subunit. Molecular cloning confirmed that the two lectin subunits are encoded by different genes. The 32 kDa subunit is apparently encoded by a single gene, whereas two highly homologous genes encode the 37 kDa subunit. A comparison of the deduced amino acid sequences of the bark lectin cDNAs and the previously described cDNA encoding the seed haemagglutinin demonstrated that they are encoded by different genes. Abbreviations: LECMAHb, cDNA clone encoding Maackia amurensis bark haemagglutinin; LECMALb, cDNA clone encoding Maackia amurensis bark leucoagglutinin; MALb, Maackia amurensis bark leucoagglutinin; MAHb, Maackia amurensis bark haemagglutinin This revised version was published online in November 2006 with corrections to the Cover Date.     

中国产家蚕抗菌肽A基因部分序列的测定

从大肠杆菌感染的家蚕蛹提取RNA,用RT-PCR方法扩增未知抗菌肽基因片段,经过克隆测序,获得了蚕抗菌肽A基因的部分片段164 bp,为制备蚕抗菌肽A基因探针,筛选基因文库打下了基础.     

Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163–168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.     

Baboon carboxylesterases 1 and 2: sequences, structures and phylogenetic relationships with human and other primate carboxylesterases

Background  Carboxylesterase (CES) is predominantly responsible for the detoxification of a wide range of drugs and narcotics, and catalyze several reactions in cholesterol and fatty acid metabolism. Studies of the genetic and biochemical properties of primate CES may contribute to an improved understanding of human disease, including atherosclerosis, obesity and drug addiction, for which non-human primates serve as useful animal models.
Methods  We cloned and sequenced baboon CES1 and CES2 and used in vitro and in silico methods to predict protein secondary and tertiary structures, and examined evolutionary relationships for these enzymes with other primate and mouse CES orthologs.
Results and Conclusions  We found that baboon CES1 and CES2 proteins retained extensive similarity with human CES1 and CES2, shared key structural features reported for human CES1, and showed family specific sequences consistent with their multimeric and monomeric subunit structures respectively.     

Isolation and characterization of a cDNA from flowers of Cynara cardunculus encoding cyprosin (an aspartic proteinase) and its use to study the organ-specific expression of cyprosin

Poly(A)⁺ RNA isolated from flower buds of Cynara cardunculus has been used to prepare a cDNA library. Screening of the cDNA after expression of cloned DNA with antibodies raised against the large subunit of cyprosin 3 resulted in the isolation of six positive clones. One of these clones (cypro1s; a 1.7 kb Eco RI fragment) codes for cyprosin. The nucleotide sequence contain a 1419 bp open reading frame coding for 473 amino acids (aa) including a putative full-length mature protein (440 aa) and a partial prosequence (33 aa). Cypro1s contains a 162 bp 3 non-coding region followed by a poly(A) tail. The deduced amino acid sequence shows high homology to other plant aspartic proteinases. The homology to mammalian and microbial aspartic proteinases is somewhat lower. Plant aspartic proteinases contain an insert of around 100 aa. We are modelling where this plant-specific insert will appear in the structure of cyprosin. Using cypro1s as a probe in northern blot analysis, the expression of cyprosin in developing flowers and other tissues has been studied. The signal on the northern blot increased for RNA samples from early (flower buds 6 mm in length) to later stages of floral development (flower buds up to 40 mm in length). In late stages of floral development (open flowers 50 mm in length and styles from such flowers) no hybridization signal was visualized showing that the synthesis of mRNA encoding the cyprosin starts in early stages of floral development and switches off at maturation of the flower. Southern blot analysis of genomic DNA showed 4–5 strong hybridizing bands and several minor bands indicating that the cyprosin genes are organized as a multi-gene family in C. cardunculus.     

甜瓜鲨烯合酶基因克隆及酶分子结构分析

葫芦素类是主要分布于葫芦科植物中具有多种医药活性的四环三萜类化合物,目前药用葫芦素原料主要从甜瓜蒂中提取。该研究从甜瓜中克隆葫芦素类合成关键酶——鲨烯合酶(SQS)的基因,并对其序列进行了生物信息学分析。结果表明:DNA测序和BLASTRefSeqGene分析表明,克隆的甜瓜SQS基因片段具有完整的该酶基因开放阅读框架(ORF)序列。ORF分析显示,甜瓜SQS由417氨基酸残基构成,等电点为7.56。对推衍的甜瓜SQS氨基酸序列分析结果提示,该酶二级结构以α螺旋为主。结构域预测结果表明,SQS属于异戊二烯合酶家族,具有法呢酰基二磷酸及镁离子的结合位点。三级结构预测提示,甜瓜SQS为单体酶,其活性中心主要由几个α螺旋围绕形成的穴状结构。磷酸化位点分析显示,S~(48)处于酶活性中心相关~(47)VSRSF~(52)的模体中,而S~(196)是正选择位点,提示这两处磷酸化位点可能是甜瓜SQS酶活性调节的关键部位。以甜瓜SQS基因ORF序列构建系统发生树的系统发生分类结果与形态学分类结果一致。该研究结果为葫芦素类的生物合成调控研究提供了新的线索和实验依据。     

Vaporization of Inorganic Mercury by Cell-free Extracts of Drug Resistant Escherichia coli

The cDNAs encoding venom phospholipase A₂ (PLA₂) inhibitors (PLIs), named Protobothrops elegans (Pe)γPLI-A, PeγPLI-B, PeαPLI-A, and PeαPLI-B, were cloned from the P. elegans liver cDNA library. They were further divided into several constituents due to nucleotide substitutions in their open reading frames. For PeαPLI-A, two constituents, PeαPLI-A_a and PeαPLI-A_b, were identified due to three nonsynonymous substitutions in exon 3. Far-western blot and mass-spectrometry analysis of the P. elegans serum proteins showed the presence of γPLIs, and αPLIs, which can bind venom PLA₂s. In αPLIs from Protobothrops sera, A or B subtype-specific amino acid substitutions are concentrated only in exon 3. A comparison of γPLIs showed that γPLI-As are conserved and γPLI-Bs diversified. Mathematical analysis of the nucleotide sequences of Protobothrops γPLI-B cDNAs revealed that the particular loops in the three-finger motifs diversified by accelerated evolution. Such evolutionary features should have made serum PLIs acquire their respective inhibitory activities to adapt to venom PLA₂ isozymes.     

大花杓兰亲环素基因的克隆及生物信息学分析

亲环素是一个多基因家族,在植物生命活动中发挥着重要的作用。该研究以大花杓兰(Cypripedium macranthum)为材料,采用RT-PCR技术克隆到1个亲环素基因(CyP),并对其进行生物信息学分析。结果表明:大花杓兰CyP基因的开放阅读框序列为525 bp,命名为CmCyP(GenBank登录号为MH411125),编码174个氨基酸。预测CmCyP蛋白是一个位于细胞质、相对分子量约为18 kD、理论pI为8.73、无信号肽、跨膜结构域的亲水性蛋白质。磷酸化和糖基化位点预测分析发现,CmCyP蛋白存在18个潜在的磷酸化位点和2个潜在的糖基化位点。蛋白保守结构域预测分析发现,CmCyP蛋白包含一个高度保守的肽脯氨酰顺反异构酶结构域,属于单结构域亲环素。对二级结构进行预测分析发现,CmCyP蛋白中存在无规卷曲70个、延伸链56个、α-螺旋23个、β-折叠25个,这4种结构元件在三级结构中也有体现。系统进化树结果显示,大花杓兰CmCyP蛋白与铁皮石斛(Dendrobium catenatum)和万带兰(Vanda hybrid cultivar)的CyP蛋白的亲缘关系较近。该研究首次克隆了大花杓兰亲环素基因(CmCyP),为进一步探讨CmCyP基因的生物学功能奠定了基础。     

Comparison of protein structure and genomic structure of human, rabbit, and limulus C-reactive proteins: Possible implications for function and evolution

The primary structures of human, rabbit, and Limulus C-reactive proteins (CRPs) have been compared by a computer program. Based on these data, a PAMs matrix (accepted point mutation per 100 residues) was constructed to generate topologies for the three proteins. Five trees with the shortest absolute length were generated, but only one positive tree was found. Using the relatively well-established distance between human and rabbit of 150 million years, we calculate that human and Limulus CRPs diverged at least 500 million years ago. The data indicate that the amino acid sequence indentity between Limulus CRPs and their mammalian counterparts is about 25%, strongly suggesting that human CRP, rabbit CRP, and Limulus CRPs share common ancestral genes. There are two highly conserved regions in the primary structures among the CRPs. Residues 52–67 in Limulus CRP and residues 51–66 in human CRP show identity in 10 of 16 positions, with 3 additional conservative replacements. This region of the molecule is thought to be involved in the binding of phosphorylcholine ligand. Residues 139–153 in Limulus CRP and residues 133–147 in human CRP show identity in 9 of 15 positions, with 5 additional conservative replacements. The biological function of this stretch of amino acid sequence is thought to be associated with the CA²⁺ binding of the CRPs.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Amino acid sequences of pilins from serologically distinct strains ofBacteroides nodosus

Amino acid sequences of pilin from a strain of Bacteroides nodosus from serogroup B (234) and serogroup C (217) were determined. The amino-terminal N-methylphenlalanine residue of both proteins was followed by a hydrophobic sequence of 30 residues closely related to the N-terminal sequence of other pili having an amino-terminal residue of N-methylphenylalanine. These data lend support to the hypothesis that in pilins of this type, the amino-terminal sequence functions as a transport signal necessary for pilin to reach its external environment, as well as promoting intersubunit interactions for muintenance of the structural integrity of the pilus. Two hydrophilic hypervariable regions can be discerned across the pilin sequences, indicating possible locations of antigenic domains.     

独行菜LaMCT基因的克隆、序列分析与原核表达

强心苷作为药用植物独行菜( Lepidium apetalum)的活性成分,其化学和药理学研究已有良好的基础,但其生物合成途径目前仍不清楚。该研究以独行菜幼苗为材料,通过分析独行菜转录组数据,设计特异性引物,PCR扩增得到了强心苷生物合成MEP途径的关键酶2-C-甲基赤藓醇-4-磷酸胞苷酰转移酶( MCT)基因的开放阅读框( ORF),命名为LaMCT( Genbank注册号KT832554),并进行序列分析和原核表达。序列分析结果表明：LaMCT基因ORF全长为912 bp,编码304个氨基酸。亚细胞定位和保守结构域分析结果表明：LaMCT蛋白位于叶绿体中,不含信号肽,没有跨膜区,含有类异戊二烯合成酶保守结构域( isoprenoid synthase domain)。系统进化树结果表明：LaMCT蛋白与拟南芥的MCT蛋白具有94％的序列相似性,亲缘关系较近。通过构建pET-32a-LaMCT原核表达载体,成功在大肠杆菌BL21( DE3)菌株中诱导表达LaMCT重组蛋白,并得到了纯化的LaMCT重组蛋白。该研究首次从独行菜中克隆了LaMCT基因,建立其稳定的原核表达体系,为LaMCT蛋白抗体的制备以及研究LaMCT基因在独行菜强心苷类化合物生物合成途径中的功能奠定了基础。     

Molecular cloning of mannose-binding lectins from Clivia miniata

Screening of a cDNA library constructed from total RNA isolated from young developing ovaries of Clivia miniata Regel with the amaryllis lectin cDNA clone resulted in the isolation of four different isolectin clones which clearly differ from each other in their nucleotide sequences and hence also in their deduced amino acid sequences. Apparently the lectin is translated from an mRNA of ca. 800 nucleotides encoding a precursor polypeptide of 163 amino acids. Northern blot analysis of total RNA isolated from different tissues of Clivia miniata has shown that the lectin is expressed in most plant tissues with very high lectin mRNA concentrations in the ovary and the seed endosperm.