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author:("Xu, deqing")
1.  Novel Family of Insect Salivary Inhibitors Blocks Contact Pathway Activation by Binding to Polyphosphate, Heparin, and Dextran Sulfate 
Polyphosphate and heparin are anionic polymers released by activated mast cells and platelets that are known to stimulate the contact pathway of coagulation. These polymers promote both the autoactivation of factor XII and the assembly of complexes containing factor XI, prekallikrein, and high-molecular-weight kininogen. We are searching for salivary proteins from blood-feeding insects that counteract the effect of procoagulant and proinflammatory factors in the host, including elements of the contact pathway.
Approach and Results
Here, we evaluate the ability of the sand fly salivary proteins, PdSP15a and PdSP15b, to inhibit the contact pathway by disrupting binding of its components to anionic polymers. We attempt to demonstrate binding of the proteins to polyphosphate, heparin, and dextran sulfate. We also evaluate the effect of this binding on contact pathway reactions. We also set out to determine the x-ray crystal structure of PdSP15b and examine the determinants of relevant molecular interactions. Both proteins bind polyphosphate, heparin, and dextran sulfate with high affinity. Through this mechanism they inhibit the autoactivation of factor XII and factor XI, the reciprocal activation of factor XII and prekallikrein, the activation of factor XI by thrombin and factor XIIa, the cleavage of high-molecular-weight kininogen in plasma, and plasma extravasation induced by polyphosphate. The crystal structure of PdSP15b contains an amphipathic helix studded with basic side chains that forms the likely interaction surface.
The results of these studies indicate that the binding of anionic polymers by salivary proteins is used by blood feeders as an antihemostatic/anti-inflammatory mechanism.
PMCID: PMC4191670  PMID: 24092749
blood coagulation factor inhibitors; bradykinin; factor XI; factor XII; inflammation; kallikreins; leishmania
2.  Examination of the Ligand-Binding and Enzymatic Properties of a Bilin-Binding Protein from the Poisonous Caterpillar Lonomia obliqua 
PLoS ONE  2014;9(6):e95424.
The bilin-binding proteins (BBP) from lepidopteran insects are members of the lipocalin family of proteins and play a special role in pigmentation through the binding of biliverdin IXγ. Lopap, a BBP-like protein from the venom of the toxic caterpillar Lonomia obliqua has been reported to act as a serine protease that activates the coagulation proenzyme prothrombin. Here we show that BBPLo, a variant of lopap from the same organism binds biliverdin IXγ, forming a complex that is spectrally identical with previously described BBP proteins. Although BBPLo is nearly identical in sequence to lopap, no prothrombinase activity was detected in our recombinant preparations using reconstituted systems containing coagulation factors Xa and Va, as well as anionic phospholipids. In addition to biliverdin, BBPLo was found to form a 1∶1 complex with heme prompting us to examine whether the unusual biliverdin IXγ ligand of BBPs forms as a result of oxidation of bound heme in situ rather than by a conventional heme oxygenase. Using ascorbate or a NADPH+-ferredoxin reductase-ferredoxin system as a source of reducing equivalents, spectral changes are seen that suggest an initial reduction of heme to the Fe(II) state and formation of an oxyferrous complex. The complex then disappears and a product identified as a 5-coordinate carbonyl complex of verdoheme, an intermediate in the biosynthesis of biliverdin, is formed. However, further reaction to form biliverdin was not observed, making it unlikely that biliverdin IXγ is formed by this pathway.
PMCID: PMC4074040  PMID: 24972000
3.  Mutations in the Homeodomain of HOXD13 Cause Syndactyly Type 1-c in Two Chinese Families 
PLoS ONE  2014;9(5):e96192.
Syndactyly type 1 (SD1) is an autosomal dominant limb malformation characterized in its classical form by complete or partial webbing between the third and fourth fingers and/or the second and third toes. Its four subtypes (a, b, c, and d) are defined based on variable phenotypes, but the responsible gene is yet to be identified. SD1-a has been mapped to chromosome 3p21.31 and SD1-b to 2q34–q36. SD1-c and SD1-d are very rare and, to our knowledge, no gene loci have been identified.
Methods and Results
In two Chinese families with SD1-c, linkage and haplotype analyses mapped the disease locus to 2q31-2q32. Copy number variation (CNV) analysis, using array-based comparative genomic hybridization (array CGH), excluded the possibility of microdeletion or microduplication. Sequence analyses of related syndactyly genes in this region identified c.917G>A (p.R306Q) in the homeodomain of HOXD13 in family A. Analysis on family B identified the mutation c.916C>G (p.R306G) and therefore confirmed the genetic homogeneity. Luciferase assays indicated that these two mutations affected the transcriptional activation ability of HOXD13. The spectrum of HOXD13 mutations suggested a close genotype-phenotype correlation between the different types of HOXD13-Syndactyly. Overlaps of the various phenotypes were found both among and within families carrying the HOXD13 mutation.
Mutations (p.R306Q and p.R306G) in the homeodomain of HOXD13 cause SD1-c. There are affinities between SD1-c and synpolydactyly. Different limb malformations due to distinct classes of HOXD13 mutations should be considered as a continuum of phenotypes and further classification of syndactyly should be done based on phenotype and genotype.
PMCID: PMC4006867  PMID: 24789103
4.  Structure and ligand-binding properties of the biogenic amine-binding protein from the saliva of a blood-feeding insect vector of Trypanosoma cruzi  
Biogenic amine-binding proteins mediate the anti-inflammatory and antihemostatic activities of blood-feeding insect saliva. The structure of the amine-binding protein from R. prolixus reveals the interaction of biogenic amine ligands with the protein.
Proteins that bind small-molecule mediators of inflammation and hemostasis are essential for blood-feeding by arthropod vectors of infectious disease. In ticks and triatomine insects, the lipocalin protein family is greatly expanded and members have been shown to bind biogenic amines, eicosanoids and ADP. These compounds are potent mediators of platelet activation, inflammation and vascular tone. In this paper, the structure of the amine-binding protein (ABP) from Rhodnius prolixus, a vector of the trypanosome that causes Chagas disease, is described. ABP binds the biogenic amines serotonin and norepinephrine with high affinity. A complex with tryptamine shows the presence of a binding site for a single ligand molecule in the central cavity of the β-barrel structure. The cavity contains significant additional volume, suggesting that this protein may have evolved from the related nitrophorin proteins, which bind a much larger heme ligand in the central cavity.
PMCID: PMC3532134  PMID: 23275168
lipocalins; Rhodnius prolixus; Triatominae; serotonin; norepinephrine; tryptamine; nitrophorin
5.  A novel family of RGD-containing disintegrin (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets integrins αIIbβ3 and αVβ3 and inhibits platelet aggregation and angiogenesis 
Thrombosis and haemostasis  2011;105(6):1032-1045.
A novel family of RGD-containing molecule (Tablysin-15) has been molecularly characterized from the salivary gland of the hematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity for platelet αIIbβ3 and endothelial cell αvβ3 integrins, but not for α5β1 or α2β1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~ 1 nM) and marginally to fibronectin (IC50 ~ 1 µM), but not to collagen. It also inhibits FGF-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilized fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbβ3 binds to Tablysin-15. Moreover, immobilized Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbβ3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow, without affecting platelet adhesion to collagen fibrils. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbβ3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.
PMCID: PMC3499621  PMID: 21475772
hematophagy; blood-sucking; disintegrin; thrombosis; sialogenins
6.  Purification and Characterization of Two New Allergens from the Venom of Vespa magnifica 
PLoS ONE  2012;7(2):e31920.
Due to poor diagnostic facilities and a lack of medical alertness, allergy to Vespa wasps may be underestimated. Few allergens have been identified from Vespa wasps.
Possible native allergen proteins were purified from the wasp venoms (WV) (Vespa magnifica Smith) by gel filtration, ion exchange chromatography, respectively. Their sequences were determined by Edman degradation and cDNA cloning. Their allergenicities were assayed by enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, and skin prick tests (SPTs). Their cross allergencities with Tab y 2 and Tab y 5 purified from the horsefly (Tabanus yao Macquart) were also determined. Two native allergens were identified from the WV, respectively. They are a 25-KDa antigen 5 protein (Ag5) (Vesp ma 5) and a 35-KDa hyaluronidase (Vesp ma 2). They represented major allergens in Vespa magnifica by immunoblots and SPTs. ELISA inhibition of pooled sera IgE reactivity to both the WV and the horsefly salivary gland extracts (HSGE) using four purified allergens (Vesp ma 2, Vesp ma 5 and previously purified Tab y 2 and Tab y 5) was significant. Their cross allergenicities were confirmed by ELISA-IT, immunoblots, and SPTs. They represented the cross reactive allergens from wasp and horsefly and proved the so called wasp-horsefly syndrome.
PMCID: PMC3288059  PMID: 22384100
7.  Association between Polymorphisms in the Promoter Regions of Matrix Metalloproteinases (MMPs) and Risk of Cancer Metastasis: A Meta-Analysis 
PLoS ONE  2012;7(2):e31251.
A variety of studies have evaluated the associations between polymorphisms in the promoter regions of Matrix metalloproteinases (MMPs) and cancer metastasis. However, the results remain inconclusive. To better understand the roles of MMP polymorphisms in metastasis, we conducted a comprehensive meta-analysis.
Electronic databases were searched (from January 2000 to June 2011) for any MMP genetic association studies in metastasis. Overall and subgroup analyses were performed. Odds ratio (OR) and 95% confidence interval (CI) were used to evaluate the associations between MMP polymorphisms and metastasis. Statistical analysis was performed with Review Manager 5.0 and STATA11.0.
Thirty-three studies addressing five MMP polymorphisms were analyzed among 10,516 cancer cases (4,059 metastasis-positive cases and 6,457 metastasis-negative cases). For MMP1 (−1607)1G/2G, genotype 2G/2G increased the overall risk of metastasis under the recessive model (OR = 1.44, 95% CI = 1.05–1.98). In subgroup analysis based on cancer type, associations were found in head/neck and breast cancer under the recessive model, and also in breast cancer under the dominant model. For MMP3 (−1171) 5A/6A, the polymorphism decreased the overall risk of metastasis under two genetic models (recessive: OR = 0.80, 95%CI = 0.64–0.99, dominant: OR = 0.72, 95%CI = 0.56–0.93). The polymorphisms of MMP7 (−181) A/G and MMP9 (−1562) C/T increased metastatic risk. However, no association was observed between MMP2 (−1306) C/T and metastasis.
Our investigations demonstrate that polymorphisms in the promoter regions of MMP1, 3, 7 and 9 might be associated with metastasis in some cancers. Further studies with large sample size for MMP2 should be conducted.
PMCID: PMC3279370  PMID: 22348060
8.  Transcriptional activation of microRNA-34a by NF-kappa B in human esophageal cancer cells 
miR-34a functions as an important tumor suppressor during the process of carcinogenesis. However, the mechanism of miR-34a dysregulation in human malignancies has not been well elucidated. Our study aimed to further investigate the regulation mechanism of miR-34a.
We found that overexpression of NF-kappa B p65 subunit could increase miR-34a levels in EC109, an esophageal squamous cancer cell line, while ectopic expression of DN IkappaB leaded to a significant reduction of miR-34a expression. Bioinformatics analysis suggested three putative KB sites in promoter region of miR-34a gene. Mutation two of these KB sites impaired p65 induced miR-34a transcriptional activity. Chromatin immunoprecipitation and electrophoretic mobility shift assays both showed that NF-kappaB could specifically bind to the third KB site located in miR-34a promoter. In addition, we found that overexpression of NF-kappaB p65 could not successfully induce miR-34a expression in esophageal cancer cell lines with mutant p53 or decreased p53. Reporter assay further showed that NF-kappaB-induced miR-34a transcriptional activity was reduced by p53 impairment. Nevertheless, CHIP analysis suggested binding of NF-kappaB to miR-34a promoter was not affected in cells with mutant p53.
Our work indicates a novel mechanism of miR-34a regulation that NF-kappaB could elevate miR-34a expression levels through directly binding to its promoter. And wildtype p53 is responsible for NF-kappaB-mediated miR-34a transcriptional activity but not for NF-kappaB binding. These findings might be helpful in understanding miR-34a abnormality in human malignancies and open new perspectives for the roles of miR-34a and NF-kappaB in tumor progression.
PMCID: PMC3311059  PMID: 22292433
miR-34a; NF-kappa B; p53; gene expression regulation
9.  Facile solution growth of vertically aligned ZnO nanorods sensitized with aqueous CdS and CdSe quantum dots for photovoltaic applications 
Nanoscale Research Letters  2011;6(1):340.
Vertically aligned single crystalline ZnO nanorod arrays, approximately 3 μm in length and 50-450 nm in diameter are grown by a simple solution approach on a Zn foil substrate. CdS and CdSe colloidal quantum dots are assembled onto ZnO nanorods array using water-soluble nanocrystals capped as-synthesized with a short-chain bifuncional linker thioglycolic acid. The solar cells co-sensitized with both CdS and CdSe quantum dots demonstrate superior efficiency compared with the cells using only one type of quantum dots. A thin Al2O3 layer deposited prior to quantum dot anchoring successfully acts as a barrier inhibiting electron recombination at the Zn/ZnO/electrolyte interface, resulting in power conversion efficiency of approximately 1% with an improved fill factor of 0.55. The in situ growth of ZnO nanorod arrays in a solution containing CdSe quantum dots provides better contact between two materials resulting in enhanced open circuit voltage.
PMCID: PMC3211429  PMID: 21711865
10.  DNaseI hypersensitivity at gene-poor, FSH dystrophy-linked 4q35.2 
Nucleic Acids Research  2009;37(22):7381-7393.
A subtelomeric region, 4q35.2, is implicated in facioscapulohumeral muscular dystrophy (FSHD), a dominant disease thought to involve local pathogenic changes in chromatin. FSHD patients have too few copies of a tandem 3.3-kb repeat (D4Z4) at 4q35.2. No phenotype is associated with having few copies of an almost identical repeat at 10q26.3. Standard expression analyses have not given definitive answers as to the genes involved. To investigate the pathogenic effects of short D4Z4 arrays on gene expression in the very gene-poor 4q35.2 and to find chromatin landmarks there for transcription control, unannotated genes and chromatin structure, we mapped DNaseI-hypersensitive (DH) sites in FSHD and control myoblasts. Using custom tiling arrays (DNase-chip), we found unexpectedly many DH sites in the two large gene deserts in this 4-Mb region. One site was seen preferentially in FSHD myoblasts. Several others were mapped >0.7 Mb from genes known to be active in the muscle lineage and were also observed in cultured fibroblasts, but not in lymphoid, myeloid or hepatic cells. Their selective occurrence in cells derived from mesoderm suggests functionality. Our findings indicate that the gene desert regions of 4q35.2 may have functional significance, possibly also to FSHD, despite their paucity of known genes.
PMCID: PMC2794184  PMID: 19820107
11.  Odorranalectin Is a Small Peptide Lectin with Potential for Drug Delivery and Targeting 
PLoS ONE  2008;3(6):e2381.
Lectins are sugar-binding proteins that specifically recognize sugar complexes. Based on the specificity of protein–sugar interactions, different lectins could be used as carrier molecules to target drugs specifically to different cells which express different glycan arrays. In spite of lectin's interesting biological potential for drug targeting and delivery, a potential disadvantage of natural lectins may be large size molecules that results in immunogenicity and toxicity. Smaller peptides which can mimic the function of lectins are promising candidates for drug targeting.
Principal Findings
Small peptide with lectin-like behavior was screened from amphibian skin secretions and its structure and function were studied by NMR, NMR-titration, SPR and mutant analysis. A lectin-like peptide named odorranalectin was identified from skin secretions of Odorrana grahami. It was composed of 17 aa with a sequence of YASPKCFRYPNGVLACT. L-fucose could specifically inhibit the haemagglutination induced by odorranalectin. 125I-odorranalectin was stable in mice plasma. In experimental mouse models, odorranalectin was proved to mainly conjugate to liver, spleen and lung after i.v. administration. Odorranalectin showed extremely low toxicity and immunogenicity in mice. The small size and single disulfide bridge of odorranalectin make it easy to manipulate for developing as a drug targeting system. The cyclic peptide of odorranalectin disclosed by solution NMR study adopts a β-turn conformation stabilized by one intramolecular disulfide bond between Cys6-Cys16 and three hydrogen bonds between Phe7-Ala15, Tyr9-Val13, Tyr9-Gly12. Residues K5, C6, F7, C16 and T17 consist of the binding site of L-fucose on odorranalectin determined by NMR titration and mutant analysis. The structure of odorranalectin in bound form is more stable than in free form.
These findings identify the smallest lectin so far, and show the application potential of odorranalectin for drug delivery and targeting. It also disclosed a new strategy of amphibian anti-infection.
PMCID: PMC2440032  PMID: 18584053

Results 1-11 (11)