cDNA cloning of two novel T-superfamily conotoxins from Conus leopardus

The full-length cDNAs of two novel T-superfamily conotoxins,Lp5.1 and Lp5.2,were clonedfrom a vermivorous cone snail Conus leopardus using 3'/5'-rapid amplification of cDNA ends.The cDNA ofLp5.1 encodes a precursor of 65 residues,including a 22-residue signal peptide,a 28-residue propeptide anda 15-residue mature peptide.Lp5.1 is processed at the common signal site -X-Arg- immediately before themature peptide sequences.In the case of Lp5.2,the precursor includes a 25-residue signal peptide anda 43-residue sequence comprising the propeptide and mature peptide,which is probably cleaved to yield a29-residue propeptide and a 14-residue mature toxin.Although these two conotoxins share a similar signalsequence and a conserved disulfide pattern with the known T-superfamily,the pro-region and mature peptidesare of low identity,especially Lp5.2 with an identity as low as 10.7% compared with the reference Mr5.1a.The elucidated cDNAs of these two toxins will facilitate a better understanding of the species distribution,the sequence diversity of T-superfamily conotoxins,the special gene structure and the evolution of thesepeptides.     

Two Potent α3/5 Conotoxins from Piscivorous Conus achatinus

Every cone snail produces a mixture of different conotoxins and secretes them to immobilize their prey and predators. α3/5 Conotoxins, isolated from fish-hunting cone snails, target muscle nicotinic acetylcholine receptors. The structure and function of α3/5 conotoxin from the piscivorous Conus achatinus have not been studied. We synthesized two pentadecamer peptides, Ac 1.1 a and Ac 1.1 b, with appropriate disulfide bonding, based on cDNA sequences of α3/5 conotoxins from C. achatinus. Ac 1.1 a and Ac 1.1 b differ by only one amino acid residue. They have similar potency on blocking recombinant mouse muscle acetylcholine receptor expressed in Xenopus laevis oocytes, with IC_(50) values of 36 nM and 26 nM, respectively. For Ac 1.1b, deletion of the first three N-terminal amino acids did not change its activity, indicating that the Nterminus is not involved in the interaction with its receptor. Furthermore, our experiments indicate that both toxins strongly prefer the α1-δ subunit interface instead of the α1-γ binding site on the mouse muscle nicotinic acetylcholine receptor. These peptides provide additional tools for the study of the structure and function of nicotinic receptor.     

Screening and identification of receptor antagonist for shiga toxin from random peptides displayed on filamentous bacteriophages

The bacteriophage clones which can bind with shiga toxin B subunit (StxB) and inhibit cytotoxicity of shiga toxin were obtained by using antibody capturing method from a 15-mer random peptide library displayed on the surface of bacteriophage fd. Among them, one peptide encoded by the random DNA region of a selected bacteriophage (A12) was synthesized and tested in vitro and in vivo, where the peptide competed with the receptor of shiga toxin to bind StxB, and inhibited the cytotoxicity and enterotoxicity of shiga toxin. The peptide can also block other apparently unrelated StxB binding bacteriophage (A3), which suggests that there are overlapping StxB interaction sites for those ligands with different sequences. The results provide a demonstration of bacteriophage display to screen peptide ligands for a small and/or unable biotinylated molecule by antibodies-capturing strategy, and take the lead for the development of receptor antagonists for shiga toxin.     

Cloning and molecular characterization of a novel lectin gene from Pinellia ternata

The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.     

Antinociceptive effects of novel epibatidine analogs through activation of α4β2 nicotinic receptors

The study of α4β2 nicotinic receptors has provided new indications in the treatment of pain. Efforts have been made to explore new α4β2 nicotinic receptor agonists, including TC-2559, as antinociceptive drugs. In this study, we discovered a set of novel epibatidine analogs with strong binding affinities to the α4β2 nicotinic receptors. Among these compounds, C-159, C-163, and C-9515 attenuated formalin-induced nociceptive responses in mice; C-9515 caused the most potent analgesic effect, which was blocked by mecamylamine, a non-selective nicotinic receptor antagonist. Furthermore, C-9515 potently inhibited chronic constriction injury(CCI)-induced neuropathic pain in rats, which was sensitive to DHβE, a selective α4β2 subtype antagonist,indicating that its analgesic effect was mediated by the activation of the α4β2 nicotinic receptors. In conclusion, the epibatidine analog C-9515 was found to be a potent α4β2 nicotinic receptor agonist with potent analgesic function, which demonstrated potential for the further exploration of its druggability.     

Microfilament-binding properties of N-terminal extension of the isoform of smooth muscle long myosin light chain kinase

Myosin light chain kinases （MLCK） phosphorylate the regulatory light chain of myosin II in thick filaments and bind to F-actin-containing thin filaments with high affinity. The ability of short myosin light chain kinase （S-MLCK） to bind F-actin is structurally attributed to the DFRXXL regions in its N-terminus. The long myosin light chain kinase （L-MLCK） has two additional DFRXXL motifs and six Ig-like modules in its N-terminal extension. The six Ig-like modules are capable of binding to stress fibers independently. Our results from the imaging analysis demonstrated that the first two intact Ig-like modules （2Ig） in N-terminal extension of L-MLCK is the minimal binding module required for microfilament binding. Binding assay confirmed that F-actin was able to bind 2Ig. Stoichiometries of 2Ig peptide were similar for myofilament or pure F-actin. The binding affinities were slightly lower than 5DFRXXL peptide as reported previously. Similar to DFRXXL peptides, the 2Ig peptide also caused efficient F-actin bundle formation in vitro. In the living cell, over-expression of 2Ig fragment increased ＂spike＂-like protrusion formation with over-bundled F-actin. Our results suggest that L-MLCK may act as a potent F-actin bundling protein via its DFRXXL region and the 2Ig region, implying that L-MLCK plays a role in cytoskeleton organization.     

Anti-HIV Ⅰ/Ⅱ Activity and Molecular Cloning of a Novel Mannose/Sialic Acidbinding Lectin from Rhizome of Polygonatum cyrtonema Hua

The anti-human immunodeficiency virus （HIV） Ⅰ/Ⅱ activity of a mannose and sialic acid binding lectin isolated from rhizomes of Polygonatum cyrtonema Hua was elucidated by comparing its HIV infection inhibitory activity in MT-4 and CEM cells with that of other mannose-binding lectins （MBLs）. The anti-HIV activity of Polygonatum cyrtonema Hua lectin （PCL） was 10- to 100-fold more potent than other tested MBLs, but without significant cytotoxicity towards MT-4 or CEM cells. To amplify cDNA of PCL by 3＇/5＇-rapid amplification of cDNA ends （RACE）, the 30 amino acids of N-terminal were determined by sequencing and the degenerate oligonucleotide primers were designed. The full-length cDNA of PCL contained 693 bp with an open reading frame encoding a precursor protein of 160 amino acid residues, consisting of a 28-residue signal peptide, a 22-residue C-terminal cleavage peptide and a 110-residue mature polypeptide which contained three tandemly arranged subdomains with an obvious sequence homology to the monocot MBL. However, only one active mannose-binding site （QDNVY） was found in subdomain Ⅰ of PCL, that of subdomain Ⅱ and Ⅲ changed to HNNVY and PDNVY, respectively. There was no intron in PCL, which was in good agreement with other monocot MBLs. Molecular modeling of PCL indicated that its three-dimensional structure resembles that of the snowdrop agglutinin. By docking, an active sialic acid-binding site was found in PCL. The instabilization of translation initiation region （TIR） in mRNA of PCL benefits its high expression in rhizomes.     

Last decade update for three-finger toxins: Newly emerging structures and biological activities

Three-finger toxins(TFTs) comprise one of largest families of snake venom toxins. While they are principal to and the most toxic components of the venoms of the Elapidae snake family, their presence has also been detected in the venoms of snakes from other families. The first TFT, α-bungarotoxin, was discovered almost 50 years ago and has since been used widely as a specific marker of the α7 and muscle-type nicotinic acetylcholine receptors. To date, the number of TFT amino acid sequences deposited in the UniProt Knowledgebase free-access database is more than 700, and new members are being added constantly.Although structural variations among the TFTs are not numerous, several new structures have been discovered recently; these include the disulfide-bound dimers of TFTs and toxins with nonstandard pairing of disulfide bonds. New types of biological activities have also been demonstrated for the well-known TFTs, and research on this topic has become a hot topic of TFT studies. The classic TFTs α-bungarotoxin and α-cobratoxin, for example, have now been shown to inhibit ionotropic receptors of γ-aminobutyric acid, and some muscarinic toxins have been shown to interact with adrenoceptors. New, unexpected activities have been demonstrated for some TFTs as well, such as toxin interaction with interleukin or insulin receptors and even TFT-activated motility of sperm. This minireview provides a summarization of the data that has emerged in the last decade on the TFTs and their activities.     

Herbicidal Activity of Toxin from Fusarium avenaceum GD-2 against Wild Oats (Avena fatua L.)

The herbicidal activity potential of toxin from Fusarium avenaceum GD-2 was evaluated against wild oats (Avena fatua L.) in this study. The toxin was assayed in vitro to evaluate its inhibition against seed germination of A. fatua. The toxin of F. avenaceum GD-2 was shown to have an inhibitory effect of around 77.54% at 5 mg/mL against germination of A. fatua seeds. The inhibitory effect shown by the toxins against radicle had higher activity than plumule under the same concentration. The toxin of F. avenaceum GD-2 significantly diminished the plant length the part on the ground with various treatments when treated with the toxin under greenhouse conditions. However, there were no significantly different reductions in plant length and the weed fresh weight with different treatments. In detached leaf injection bioassay, the toxic metabolite was characterized after the culture filtrates crude extraction with petroleum ether, chloroform, ethyl acetate and n-butanol. The residues left after solvent evaporation were evaluated separately for their toxicity against the target weed. Residue (5 mg/mL) obtained from n butanol fraction showed the highest toxic activity when compared with others. Moreover, a host range experiments on the sensitivity of 10 plant species revealed that barnyard grasses and goosefeet were more sensitive to the toxin of the culture filtrate. Three herbicidal active compounds were isolated and purified from cultural filtrate with the same UV absorption peak. The recent results showed potential for the development of the toxins produced by F. avenaceum GD-2 as a bio herbicidal source to control and eliminate A. fatua weed.     

家蝇防御素基因的cDNA克隆及序列分析

Defensin is a kind of cationic.inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms.with their broad antimicrobial spectrum and strong pharmaceutical effects.antimicrobial peptides,including defensins,represent a source of novel antibiotic agents.A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDnA library of houseflies(Musca domestica) that had been challenged by E.coli and staphylococcus taincd an NH2-terminal signal sequence(1-22)followed by a propeptide and the mature peptide(53-92),The sequence identity with other insect defensin is between 51% and 73%.The mature peptide,with a predicted molecular weight of 4.0kDa,and pI of 8.69,has 1 negative charged amino acid and 4 positice ones,the putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds,Cys1-Cys4,Cys2-Cys5 and Cys3-Cys6,These results suggest that the novel full-length cDNA of the defensin gene.Denominated Mdde,has been successfully cloned from houseflies.     

Engineering human interferon α1c/86D with phage display technology

Human interferon-α1c/86D (IFNα1c/86D) was functionally displayed on the surface of the filamentous bacteriophage using a phagemid vector system (pCANTAB5E). The key amino acid residues involved in the receptor binding were further defined with phage displayed 6-mer peptide library and two neutralizing antibodies against linear epitopes on the IFN-α1b, indicating that residues 30, 33, 34, (AB-loop) and residues 124, 126, 127 (D helix, DE-loop) were more critical than the adjacent residues for recognition of receptor. In addition, a cassette mutagenesis library was generated by fully randomizing the sequence of the four positions 29, 31, 32 and 35 in AB-loop, and used to select phage-IFN variants with WISH-hased panning method. Three phage-IFN variants were isolated to possess more antiviral activity in the range of 4—16-fold than parental phage-IFN after IPTG-induced soluble expression. The results suggest that phage displayed phage-IFN α1c/86D variants with increased specific activity might be obta     

Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LCs0 value of 18.4 uM and 0.70 μM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 μM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3μM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na influx and finally cause destruction of insect cells.     

Molecular Characterization of a Dehydroascorbate Reductase from Pinus bungeana

Dehydroascorbate reductase （DHAR） plays a critical role in the ascorbate-glutathione recycling reaction for most higher plants. To date, studies on DHAR in higher plants have focused largely on Arabidopsis and agricultural plants, and there is virtually no information on the molecular characteristics of DHAR in gymnosperms. The present study reports the cloning and characteristics of a DHAR （PbDHAR） from a pine, Pinus bungeana Zucc. ex Endl. The PbDHAR gene encodes a protein of 215 amino acid residues with a calculated molecular mass of 24.26 kDa. The predicted 3-D structure of PbDHAR showed a typical glutathione S-transferase fold. Reverse transcripUon-polymerase chain reaction revealed that the PbDHAR was a constitutive expression gene in P. bungeana. The expression level of PbDHAR mRNA in P. bungeana seedlings did not show significant change under high temperature stress. The recombinant PbDHAR was overexpressed in Escherichia coil following purification with affinity chromatography. The recombinant PbDHAR exhibited enzymatic activity （19.84 i.mnol/min per mg） and high affinity （a Krn of 0.08 mM） towards the substrates dehydroascorbate （DHA）. Moreover, the recombinant PbDHAR was a thermostable enzyme, and retained 77% of its initial activity at 55℃. The present study is the first to provide a detailed molecular characterization of the DHAR in P. bungeana.     

Distribution of α-D-mannose residue on zona pellucida and its role(s) in fertilization in pigs

The present study aims to identify the distribution of α-D-mannose residues on zona pellucida (ZP) and their role(s) in fertilization in pigs. In experiment 1, in vitro matured pig oocytes were freed from cu- mulus cells and treated with fluorescein isothiocyanate-labelled Lens culinaris (FITC-LCA), a D-mannose specific binding lectin. After 30 min of treatment, LCA bound evenly throughout the ZP with strong fluorescence. In experiment 2, when LCA-treated oocytes were used for in vitro fertilization, the number of sperm bound to ZP was significantly decreased, and sperm penetration was almost com- pletely blocked. In experiment 3, polysaccharide mannan was added to the in vitro fertilization medium as a competitive inhibitor. Both the number of sperm bound to ZP and the rate of fertilized oocytes were significantly reduced in the mannan-treated group compared with the control group. In experiment 4, spermatozoa were incubated with mannan in vitro. The number of acrosome-reacted spermatozoa was evidently increased in a time-dependent manner during the incubation. These results suggest that α-D-mannose residues presenting on pig ZP might be an important component of sperm receptor and might induce sperm acrosome reaction and thus facilitate the sperm penetration into the ZP.     

Provincial distribution of three HIV-1 resistant polymorphisms (CCR5-Δ32, CCR2-64I, and SDF1-3' A) in China

CCR5-Δ2, CCR2-641, mutants in two chemokine receptors and SDF1-3' A, mutant in a ligand gene, can delay AIDS pathogenesis. The distribution of the three polymorphic loci was studied in 1 046 DNA samples from 26 provinces (cities) in China. No CCR5-Δ32 was observed. CCR2-64I and SDF1-3' A had reverse distribution cline from south to north in China, with average frequency of 20.8% and 24.8% respectively. Relative hazard was evaluated. Important information to the epidemiology of AIDS and the origin and spread of these polymorphic loci in Chinese was provided.     

DISCOVERY OF THE "PEKING MAN" AND INTERNATIONAL SCIENTIFIC COOPERATION

On October 16, 1927 Dr. Birger Bohlin, a Swedish paleontologist, recovered at Zhoukoudian in Fangshan County a hominid lower molar tooth, the first tooth of the Peking Man to have been identified on spot in the field. The late Prof. Davidson Black, a Canadian anatomist and anthropologist, made a detailed study of the tooth, together with two teeth excavated previously from the same site by the Austrian paleontologist, Otto Zdansky during the field season of 1921 and 1923. The result of the study was published in the same year of 1927, as a fascicle in volume Ⅶ of the monographic series Palaeontologica Sinica. It was in this article that the first Chinese Ape-Man was christianized as "Sinanthropus pekinensis", coauthored by Black and Zdansky,with Bohlin's lower molar as its type specimen.