寡肽转运蛋白PepT2原核表达质粒的构建及其表达初探

寡肤转运蛋白(PepT2,peptide transporter,SLC15A2)是哺乳动物体内能够转运二肤、三肽的蛋白.研究表明,一些类肽的小分子药物也是PepT2的底物,但PepT2的结构与生物学功能尚待研究.建立稳定表达PepT2的表达体系是研究PepT2的重要环节.根据GenBank中人PepT2基因序列,借助Primer5.0设计了1对寡核苷酸引物,经PCR合成长达2 190bp的目的序列,通过重组构建pET30a(+)/PepT2表达质柱,测序分析确认目的基因中的3个碱基发生突变.初步研究了pET30a(+)/PepT2在大肠杆菌BL21(DE3)pLysS中的表达,为PepT2原核表达的进一步科研和实际应用奠定了基础.     

Metabolic flux analysis of Escherichia coli K12 grown on 13C-labeled acetate and glucose using GC-MS and powerful flux calculation method

A new algorithm was developed for the estimation of the metabolic flux distribution based on GC-MS data of proteinogenic amino acids. By using a sensitive GC-MS protocol as well as by combining the global search algorithm such as the genetic algorithm with the local search algorithm such as the Levenberg-Marquardt algorithm, not only the distribution of the net fluxes in the entire network, but also certain exchange fluxes which contribute significantly to the isotopomer distribution could be quantified. This mass isotopomer analysis could identify the biochemical changes involved in the regulation where acetate or glucose was used as a main carbon source. The metabolic flux analysis clearly revealed that when the specific growth rate increased, only a slight change in flux distribution was observed for acetate metabolism, indicating that subtle regulation mechanism exists in certain key junctions of this network system. Different from acetate metabolism, when glucose was used as a carbon source, as the growth rate increased, a significant increase in relative pentose phosphate pathway (PPP) flux was observed for Escherichia coli K12 at the expense of the citric acid cycle, suggesting that when growing on glucose, the flux catalyzed by isocitrate dehydrogenase could not fully fulfill the NADPH demand for cell growth, causing the oxidative PPP to be utilized to a larger extent so as to complement the NADPH demand. The GC-MS protocol as well as the new algorithm demonstrated here proved to be a powerful tool for characterizing metabolic regulation and can be utilized for strain improvement and bioprocess optimization.     

PRIP (Phospholipase C-related but Catalytically Inactive Protein) Inhibits Exocytosis by Direct Interactions with Syntaxin 1 and SNAP-25 through Its C2 Domain

Membrane fusion for exocytosis is mediated by SNAREs, forming trans-ternary complexes to bridge vesicle and target membranes. There is an array of accessory proteins that directly interact with and regulate SNARE proteins. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. However, the roles of the C2 domain have not yet been investigated. In this study, we found that the C2 domain of PRIP directly interacts with syntaxin 1 and SNAP-25 but not with VAMP2. The C2 domain promoted PRIP to co-localize with syntaxin 1 and SNAP-25 in PC12 cells. The binding profile of the C2 domain to SNAP-25 was comparable with that of synaptotagmin I, and PRIP inhibited synaptotagmin I in binding to SNAP-25 and syntaxin 1. It was also shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K⁺-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNARE-binding, C2 domain-containing accessory proteins such as synaptotagmin I and by directly inhibiting trans-SNARE complex formation.     

PSMD: An extensive database for pan‐species microsatellite investigation and marker development

Microsatellites are widely distributed throughout nearly all genomes which have been extensively exploited as powerful genetic markers for diverse applications due to their high polymorphisms. Their length variations are involved in gene regulation and implicated in numerous genetic diseases even in cancers. Although much effort has been devoted in microsatellite database construction, the existing microsatellite databases still had some drawbacks, such as limited number of species, unfriendly export format, missing marker development, lack of compound microsatellites and absence of gene annotation, which seriously restricted researchers to perform downstream analysis. In order to overcome the above limitations, we developed PSMD (Pan‐Species Microsatellite Database, http://big.cdu.edu.cn/psmd/ ) as a web‐based database to facilitate researchers to easily identify microsatellites, exploit reliable molecular markers and compare microsatellite distribution pattern on genome‐wide scale. In current release, PSMD comprises 678,106,741 perfect microsatellites and 43,848,943 compound microsatellites from 18,408 organisms, which covered almost all species with available genomic data. In addition to interactive browse interface, PSMD also offers a flexible filter function for users to quickly gain desired microsatellites from large data sets. PSMD allows users to export GFF3 formatted file and CSV formatted statistical file for downstream analysis. We also implemented an online tool for analysing occurrence of microsatellites with user‐defined parameters. Furthermore, Primer3 was embedded to help users to design high‐quality primers with customizable settings. To our knowledge, PSMD is the most extensive resource which is likely to be adopted by scientists engaged in biological, medical, environmental and agricultural research.     

THE ULTRASTRUCTURE OF THE ENAMEL IN THE GIANT PANDA OF PLEISTOCENE

<正> Enamel ultrastructures in the molar teeth of the giant panda, including Ailuropoda microta of the Early Pleistocene, Ailuropoda melanoleuca bacont of the Middle and Late Pleistocene and a living form, Ailuropoda rnelanoleuca, have been investigated by scanning electron microscopy. Transverse and longitudinal sections of enamel were made in order co evaluate shape, size and arrangement of the prisms. The sections were etched then with 0.074 M H_3PO_4 for 30-60 sec. Our investigations have shown certain features of the enamel which allow us to recognize differences among Ailuropoda on the basis of examination of large areas of the enamel. The results are summarized below.     

Analysis of variable sites between two complete South China tiger (Panthera tigris amoyensis) mitochondrial genomes

In order to investigate the mitochondrial genome of Panthera tigris amoyensis, two South China tigers (P25 and P27) were analyzed following 15 cymt-specific primer sets. The entire mtDNA sequence was found to be 16,957 bp and 17,001 bp long for P25 and P27 respectively, and this difference in length between P25 and P27 occurred in the number of tandem repeats in the RS-3 segment of the control region. The structural characteristics of complete P. t. amoyensis mitochondrial genomes were also highly similar to those of P. uncia. Additionally, the rate of point mutation was only 0.3% and a total of 59 variable sites between P25 and P27 were found. Out of the 59 variable sites, 6 were located in 6 different tRNA genes, 6 in the 2 rRNA genes, 7 in non-coding regions (one located between tRNA-Asn and tRNA-Tyr and six in the D-loop), and 40 in 10 protein-coding genes. COI held the largest amount of variable sites (9 sites) and Cytb contained the highest variable rate (0.7%) in the complete sequences. Moreover, out of the 40 variable sites located in 10 protein-coding genes, 12 sites were nonsynonymous.     

Specificity of Pyrrolysyl-tRNA Synthetase for Pyrrolysine and Pyrrolysine Analogs

Pyrrolysine, the 22nd amino acid, is encoded by amber (TAG = UAG) codons in certain methanogenic archaea and bacteria. PylS, the pyrrolysyl-tRNA synthetase, ligates pyrrolysine to tRNA^Pyl for amber decoding as pyrrolysine. PylS and tRNA^Pyl have potential utility in making tailored recombinant proteins. Here, we probed interactions necessary for recognition of substrates by archaeal PylS via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays. Replacement of the methylpyrroline ring of pyrrolysine with cyclopentane indicated that solely hydrophobic interactions with the ring-binding pocket of PylS are sufficient for substrate recognition. However, a 100-fold increase in the specificity constant of PylS was observed with an analog, 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2Thf-lys), in which tetrahydrofuran replaced the pyrrolysine methylpyrroline ring. Other analogs in which the electronegative atom was moved to different positions suggested PylS preference for a hydrogen-bond-accepting group at the imine nitrogen position in pyrrolysine. 2Thf-lys was a preferred substrate over a commonly employed pyrrolysine analog, but the specificity constant for 2Thf-lys was 10-fold lower than for pyrrolysine itself, largely due to the change in K_m. The in vivo activity of the analogs in supporting UAG suppression in Escherichia coli bearing genes for PylS and tRNA^Pyl was similar to in vitro results, with l-pyrrolysine and 2Thf-lys supporting the highest amounts of UAG translation. Increasing concentrations of either PylS substrate resulted in a linear increase in UAG suppression, providing a facile method to assay bioactive pyrrolysine analogs. These results illustrate the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyrrolysine and provide a promising new series of easily synthesized pyrrolysine analogs that can serve as scaffolds for the introduction of novel functional groups into recombinant proteins.     

Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.     

Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination

Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination.