2-Methoxypyridine as a Thymidine Mimic in Watson–Crick Base Pairs of DNA and PNA: Synthesis,Thermal Stability,and NMR Structural Studies

The development of nucleic acid base-pair analogues that use new modes of molecular recognition is important both for fundamental research and practical applications. The goal of this study was to evaluate 2-methoxypyridine as a cationic thymidine mimic in the A–T base pair. The hypothesis was that including protonation in the Watson–Crick base pairing scheme would enhance the thermal stability of the DNA double helix without compromising the sequence selectivity. DNA and peptide nucleic acid (PNA) sequences containing the new 2-methoxypyridine nucleobase (P) were synthesized and studied by using UV thermal melting and NMR spectroscopy. Introduction of P nucleobase caused a loss of thermal stability of ≈10 °C in DNA–DNA duplexes and ≈20 °C in PNA–DNA duplexes over a range of mildly acidic to neutral pH. Despite the decrease in thermal stability, the NMR structural studies showed that P–A formed the expected protonated base pair at pH 4.3. Our study demonstrates the feasibility of cationic unnatural base pairs; however, future optimization of such analogues will be required.     

多肽固相合成缩合试剂—HBTU合成方法的改进

２－（１Ｈ－苯并三唑）－Ｎ，Ｎ，Ｎ＇，Ｎ＇－四甲基脲六氟磷酸盐最近年来发展起来的一种理想的缩合试剂，已多肽固相合成得到了广泛应。     

A comparison of biological activities of a new soya biopeptide studied in an in vitro skin equivalent model and human volunteers

During aging, the epidermis and dermis become thin and an efficient anti-aging product should be able to stimulate the metabolism of senescent fibroblast and keratinocytes, in order to increase the quantity of extra-cellular matrix components such as collagen and glycosaminoglycans. A study performed in parallel on an in vitro skin equivalent model, and in vivo, with human volunteers, demonstrated the efficacy of one specific soya biopeptide for anti-aging properties. Such a biopeptide induces a significant increase of glycosaminoglycans synthesis in vitro and in vivo after a one-month treatment. We also showed that this new cosmetic ingredient is able to stimulate favourably the collagen synthesis in vitro and in vivo. This study provided the proof for anti-aging properties of a new soya biopeptide and also validated the skin equivalent model developed for this experimentation as an alternative method to animal or human testing for some cosmetic efficacy evaluations.     

Rational design and selection of bivalent peptide ligands of thrombin incorporating P4-P1 tetrapeptide sequences: from good substrates to potent inhibitors

The tetrapeptide Phe-Asn-Pro-Arg is a structurally optimized sequence for binding to the active site of thrombin. By conjugating this tetrapeptide or some variants to a C-terminal fragment of hirudin, we were able to generate a series of new bivalent inhibitors of thrombin containing only genetically encodable natural amino acids. We found that synergistic binding to both the active site and an exosite of thrombin can be enhanced through substitutions of amino acid residues at the P3 and P3' sites of the active-site directed sequence, Phe(P4)-Xaa(P3)-Pro(P2)-Arg(P1)-Pro(P1')-Gln(P2')-Yaa(P3'). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P4, P3 and P3' sites for bivalent and optimized two-site binding. Very significantly, panning of the phage library has led to thrombin-inhibitory peptides possessing strong anti-clotting activities in the low nanomolar range and yet interfering only partially the catalytic active site of thrombin. Modes of action of the newly discovered bivalent inhibitors are rationalized in light of the allosteric properties of thrombin, especially the interplay between the proteolytic action and regulatory binding occurring at thrombin surfaces remote from the catalytic active site.     

Accepting its random coil nature allows a partial NMR assignment of the neuronal Tau protein

A combined strategy to obtain a partial NMR assignment of the neuronal Tau protein is presented. Confronted with the extreme spectral degeneracy that the spectrum of this 441 amino acid long unstructured protein presents, we have introduced a graphical procedure based on residue type-specific product planes. Combining this strategy with the search for pairwise motifs, and combining the spectra of different Tau isoforms and even of peptides derived from the native sequence, we arrive at a partial assignment that is sufficient to map the interactions of Tau with its molecular partners. The obtained assignments equally confirm the absence of regular secondary structure in the isolated protein.     

Identification and Cloning of a Pheromone-Binding Protein from the Oriental Beetle, Exomala orientalis

We have identified and cloned a pheromone-binding protein (EoriPBP) from the Japanese and American populations of the Oriental beetle, Exomala orientalis (Coleoptera: Scarabaeidae). The protein showed more than 90% amino acid identity to the previously identified pheromone-binding proteins from Popillia japonica (PjapPBP) and Anomala osakana (AosaPBP), as well as to one of the odorant-binding proteins from Phyllopertha diversa (PdivOBP1). EoriPBP has 116 amino acids, with a calculated molecular mass of 12,981 Da, pI of 4.3, and six highly conserved cysteine residues. 5-RACE amplifications led to the characterization of a signal peptide with 19 amino acids. The signal peptide showed high amino acid identity to the signal peptide for AosaPBP. Comparison of the amino acid sequences of the PBPs involved in the detection of similar ligands, i.e., monounsaturated lactones and ketone, suggests that the most variable residues among the PBPs from E. orientalis, P. japonica, and A. osakana are probably the most discriminating residues. As with the pheromone-binding protein from Bombyx mori, the residues at positions 61, 64, 71, and 82 in EoriPBP, PajpPBP, and AosaPBP, which are either valine, leucine, isoleucine, or methionine, are likely to be specificity determinants.     

Eetopie Bone Formation in vivo Indueed by a Novel Synthetie Peptide Derived from BMP-2 Using Porous Collagen Scaffolds

To investigate the osteoinductive and ectopicly osteogenic effects of a novel peptide P24 derived from bone morphogenetic protein 2 （BMP2）, biodegradable collagen scaffolds （CS） were used to load BMP-2-derived peptide solutions with different concentrations （0.4 mg peptide/CS, 0.1 mg peptide/CS and pure CS, respectively）, and the implants were implanted into muscular pockets on the back of Wistar rats. Radiographs and histological analysis were performed to evaluate the ectopic bone effects. Active ectopic bone formation was seen in both groups containing the peptide at different concentration （0.4 mg and 0.1 mg）, whereas no bone formation and only fibrous tissue was seen in the pure CS group. The new bone formation induced by the peptide P24 displayed a dose-dependent and time-dependent efficiency. The new bone formation in the 0.4 mg peptide/CS group significantly increased than that of the 0.1 mg peptide/CS group. This novel BMP-2-derived peptide had excellent osteoinductive and ectopicly osteogenic properties which were similar to those of BMP2.     

A proteomic analysis of green and white sturgeon larvae exposed to heat stress and selenium

Temperature and selenium are two environmental parameters that potentially affect reproduction and stock recruitment of sturgeon in the San Francisco Bay/Delta Estuary. To identify proteins whose expression is modified by these environmental stressors, we performed a proteomic analysis on larval green and white sturgeons exposed to 18 or 26 °C and micro-injected with Seleno-L-Methionine to reach 8 µg g⁻¹ selenium body burden, with L-Methionine as a control. Selenium and high temperature induced mortalities and abnormal morphologies in both species, with a higher mortality in green sturgeon. Larval proteins were separated by two-dimensional gel electrophoresis and differential abundances were detected following spot quantitation and hierarchical cluster analysis. In green sturgeon, 34 of 551 protein spots detected on gels showed a variation in abundance whereas in white sturgeon only 9 of 580 protein spots were differentially expressed (P < 0.01). Gel replicates were first grouped according to heat treatment. Fifteen of these spots were identified using MALDI TOF/TOF mass spectrometry. Proteins involved in protein folding, protein synthesis, protein degradation, ATP supply and structural proteins changed in abundance in response to heat and/or selenium. 40S ribosomal protein SA, FK506-binding protein 10, 65 kDa regulatory subunit A of protein phosphatase 2, protein disulfide isomerase, stress-induced-phosphoprotein 1, suppression of tumorigenicity 13 and collagen type II alpha 1, were differentially expressed in high temperature treatment only. Serine/arginine repetitive matrix protein 1, creatine kinase, serine peptidase inhibitor Kazal type 5 and HSP90 were sensitive to combined temperature and selenium exposure. Valosin-containing protein, a protein involved in aggresome formation and in protein quality control decreased more than 50% in response to selenium treatment. Potential use of such proteins as biomarkers of environmental stressors in larval sturgeons could indicate early warning signals preceding population decline.