Biomedical subjects

Wan-Yu Wang

Publications and source records attributed to Wan-Yu Wang.

At least 19 recordsLinked to original sources

PubMed2006

Purification, characterization and cytokine release function of a novel Arg-49 phospholipase A(2) from the venom of Protobothrops mucrosquamatus.

Group IIA phospholipase A(2) (PLA(2)) are major components in Viperidae/Crotalidae venom. In the present study, a novel PLA(2) named promutoxin with Arg at the site 49 has been purified from the venom of Protobothrops mucrosquamatus by chromatography. It consists of 122 amino acid residues with a molecular mass of 13,656 Da assessed by MALDI-TOF. It has the structural features of snake venom group IIA PLA(2)s, but has no PLA(2) enzymatic activity. Promutoxin shows higher amino acid sequence identity to the K49 PLA(2)s (72-95%) than to D49 PLA(2)s (52-58%). Promutoxin exhibits potent myotoxicity in the animal model with as little as 1 microg of promutoxin causing myonecrosis and myoedema in the gastrocnemius muscle of mice. Promutoxin is also able to stimulate the release of IL-12, TNFalpha, IL-6 and IL-1beta from human monocytes, and induce IL-2, TNFalpha and IL-6 release from T cells, indicating that this snake venom group IIA PLA(2) is actively involved in the inflammatory process in man caused by snake venom poisoning.

Amino Acid Sequence↗

PubMed2005

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2.

A novel phospholipase A2 (PLA2) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C18 chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A2s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A2s are distinct from the other subgroups (D49 PLA2, S49 PLA2 and K49 PLA2) and represent a unique subgroup of snake venom group II PLA2, named N49 PLA2 subgroup.

Amino Acid Sequence↗

PubMed2005

Characterization and molecular cloning of dabocetin, a potent antiplatelet C-type lectin-like protein from Daboia russellii siamensis venom.

A novel C-type lectin-like protein, dabocetin, was purified from Daboia russellii siamensis venom. On SDS-polyacrylamide gel electrophoresis, it showed a single band with an apparent molecular weight of 28 kDa and two distinct bands with the apparent molecular weights of 15.0 kDa and 14.5 kDa under non-reducing and reducing conditions, respectively. cDNA clones containing the coding sequences for dabocetin alpha and beta subunits were isolated and sequenced. The deduced protein sequences of both subunits were confirmed by N-terminal amino acid sequencing and trypsin-digested peptide mass fingerprinting. Dabocetin did not induce platelet aggregation in platelet-rich plasma. It also had little effect on the platelet aggregation induced by ADP, TMVA or stejnulxin. Whereas, dabocetin inhibited ristocetin-induced platelet agglutination in platelet-rich plasma in a dose-dependent manner with an IC50 value of 0.35 microM. Flow cytometry analysis showed that dabocetin significantly inhibited mAb SZ2 binding to platelet membrane glycoprotein Ib alpha, indicating that platelet membrane glycoprotein Ib is involved in the inhibitory effect of dabocetin on ristocetin-induced platelet agglutination.

Adenosine Diphosphate↗

PubMed2005

Potent histamine-releasing activity of atrahagin, a novel snake venom metalloproteinase.

Poisonous snakebite wound is a popular disease worldwide. However, the pathogenesis remains unclear. In the present study, a novel metalloproteinase atrahagin in Chinese cobra (Naja atra) snake venom was purified, using heparin-sepharose followed by Superdex 75 gel filtration chromatography. Apart from its alpha-fibrinogenase activity, atrahagin potently activated human colon, lung and tonsil mast cells with the net histamine release being 25.9+/-4.4, 17.0+/-1.9, 13.2+/-3.6%, respectively. Time course studies revealed that the peak histamine release induced by atrahagin occurred at 12, 12 and 8 min following incubation of the enzyme with colon, lung and tonsil mast cells, respectively. The response of mast cells to atrahagin was abolished by preincubation of the cells with metabolic inhibitors or pertussis toxin, and by removal of Ca2+ and Mg2+ from the challenge buffer. In conclusion, activation of human mast cells by atrahagin indicated that the enzyme might contribute to the pathogenesis of snakebite wound.

Animals↗

PubMed2005

Neuroprotective flavonoids from Flemingia macrophylla.

Using an Abeta-induced neurotoxicity blocking assay to direct fractionation, three new flavonoids, fleminginin (1), flemingichromone (2), and flemingichalcone (3), and twenty known compounds were isolated from the active fractions of the aerial parts of Flemingia macrophylla. The structures of 1 - 3 were elucidated on the basis of spectroscopic data. When tested for neuroprotective activity, compound 2, osajin ( 4), 5,7,4'-trihydroxy-6,8-diprenylisoflavone (5), 5,7,4'-trihydroxy-6,3'-diprenylisoflavone (6), and aureole (7) protected neuronal cells from Abeta-induced damage with EC50 values of 31.43 +/- 3.16, 5.01 +/- 1.28, 11.25 +/-1.51, 4.47 +/- 0.65, 12.09 +/- 2.55 microM, respectively.

Alzheimer Disease↗

PubMed2004

A novel high molecular weight metalloproteinase cleaves fragment F1 of activated human prothrombin.

A hemorrhagic proteinase, jerdohagin, was purified from Trimeresurus jerdonii venom by gel filtration and ion-exchange chromatographies. It was a single chain polypeptide with an apparent molecular weight of 96 kDa as estimated by SDS-PAGE under the non-reducing and reducing conditions. Internal peptide sequencing indicated that it consisted of metalloproteinase, disintegrin-like and cysteine-rich domains and belonged to the class III snake venom metalloproteinases (class P-III SVMPs). Like other typical metalloproteinases, hemorrhagic activities of jerdohagin were completely inhibited by EDTA, but not by PMSF. Jerdohagin preferentially degraded alpha-chain of human fibrinogen. Interestingly, jerdohagin did not activate human prothrombin, whereas it cleaved human prothrombin and fragment F1 of activated human prothrombin.

Amino Acid Sequence↗

PubMed2004

Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom.

A chymotrypsin inhibitor, designated NA-CI, was isolated from the venom of the Chinese cobra Naja atra by three-step chromatography. It inhibited bovine alpha-chymotrypsin with a Ki of 25 nM. The molecular mass of NA-CI was determined to be 6403.8 Da by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) analysis. The complete amino acid sequence was determined after digestion of S-carboxymethylated inhibitor with Staphylococcus aureus V8 protease and porcine trypsin. NA-CI was a single polypeptide chain composed of 57 amino acid residues. The main contact site with the protease (P1) has a Phe, showing the specificity of the inhibitor. NA-CI shared great similarity with the chymotrypsin inhibitor from Naja naja venom (identities=89.5%) and other snake venom protease inhibitors.

Amino Acid Sequence↗

PubMed2004

A novel disintegrin, jerdonatin, inhibits platelet aggregation and sperm-egg binding.

A novel disintegrin, jerdonatin, was purified to homogeneity from Trimeresurus jerdonii venom by gel filtration and reversed-phase high-pressure liquid chromatography. We isolated the cDNA encoding jerdonatin from the snake venom gland. Jerdonatin cDNA precursor encoded pre-peptide, metalloprotease and disintegrin domain. Jerdonatin is composed of 72 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonatin was determined to be 8011 Da by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Jerdonatin inhibited ADP- and collagen-induced human platelet aggregation with IC50 of 123 and 135 nM, respectively. We also investigated the effect of jerdonatin on the binding of B6D2F1 hybrid mice spermatozoa to mice zona-free eggs and their subsequent fusion. Jerdonatin significantly inhibited sperm-egg binding in a concentration-dependent manner, but had no effect on the fusion of sperm-egg. These results indicate that integrins on the egg play a role in mammalian fertilization.

Amino Acid Sequence↗

PubMed2004

Purification, cloning and biological characterization of a novel disintegrin from Trimeresurus jerdonii venom.

A novel disintegrin, jerdonin, was purified from the Trimeresurus jerdonii venom by means of gel filtration and reverse phase high pressure liquid chromatography. Its coding cDNA was also isolated from the venom gland. The jerdonin coding cDNA is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. From the deduced amino acid sequence, jerdonin is composed of 71 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonin was determined to be 7483Da by matrix-assisted laser desorption ionization time of flight mass spectrometry. Jerdonin inhibited ADP- and collagen-induced human platelet aggregation with IC(50) of 220 and 240 nM, respectively. In vivo, jerdonin inhibited the growth of subcutaneously inoculated B16 solid tumor in C57BL/6 mice and improved the survival time of the tumor-bearing mice.

Amino Acid Sequence↗

PubMed2004

TMVA, a snake C-type lectin-like protein from Trimeresurus mucrosquamatus venom, activates platelet via GPIb.

TMVA is a C-type lectin-like protein with potent platelet activating activity from Trimeresurus mucrosquamatus venom. In the absence of von Willebrand factor (vWF), TMVA dose-dependently induced aggregation of washed platelets. Anti-GP Ib monoclonal antibodies (mAbs), HIP1, specifically inhibited TMVA-induced aggregation in a dose-dependent manner. The aggregation was also inhibited by mAb P2 (an anti-GP IIb mAb). Flow cytometric analysis revealed that FITC-TMVA bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by HIP1 in a dose-dependent manner. Flow cytometric analysis showed that TMVA did not bind to platelet GPIX, GPIIb, GPIIIa, GPIa, GPIIa and GPIV. Moreover, the platelet aggregation induced by TMVA was partially inhibited when platelet was pretreated with mocarhagin, a snake venom protease that specifically cleaves human GPIb. These results suggest that TMVA is a strong platelet agonist via GPIb and it might have multiple functional binding-sites on GPIb molecule or on other unknown receptor.

Animals↗

PubMed2004

TMVA, a novel GPIb-binding protein, significantly prevents platelet microthrombi formation and prolongs discordant cardiac xenograft survival.

In xenotransplantation, donor endothelium is the first target of immunological attack. Activation of the endothelial cell by preformed natural antibodies leads to platelet binding via the interaction of the glycoprotein (GP) Ib and von Willebrand factor (vWF). TMVA is a novel GPIb-binding protein purified from the venom of Trimeresurus mucrosquamatus. In this study, the inhibitory effect of TMVA on platelet aggregation in rats and the effect on discordant guinea pig-to-rat cardiac xenograft survival were investigated. Three doses (8, 20 or 40 microg/kg) of TMVA were infused intravenously to 30 rats respectively. Platelet aggregation rate was assayed 0.5, 12, and 24 h after TMVA administration. Wister rats underwent guinea pig cardiac cervical heterotopic transplantation using single dosing of TMVA (20 microg/kg, i.v., 0.5 h before reperfusion). Additionally, levels of TXB(2) and 6-keto-PGF(1alpha) within rejected graft tissues were determined by radioimmunoassay. Treatment with TMVA at a dose of 20 or 40 microg/kg resulted in complete inhibition of platelet aggregation 0.5 h after TMVA administration. Rats receiving guinea pig cardiac xenografts after TMVA therapy had significantly prolonged xenograft survival. Histologic and immunopathologic analysis of cardiac xenografts in TMVA treatment group showed no intragraft platelet microthrombi formation and fibrin deposition. Additionally, the ratio of 6-keto-PGF(1alpha) to TXB(2) in TMVA treatment group was significantly higher than those in control group. We conclude that the use of this novel GPIb-binding protein was very effective in preventing platelet microthrombi formation and fibrin deposition in a guinea pig-to-rat model and resulted in prolongation of xenograft survival. The increased ratio of PGI(2)/TXA(2) in TMVA treatment group may protect xenografts from the endothelial cell activation and contribute to the prolongation of xenograft survival.

Adenosine Diphosphate↗

PubMed2004

Improved suppression of circulating complement does not block acute vascular rejection of pig-to-rhesus monkey cardiac transplants.

At present, acute vascular rejection (AVR) remains a primary obstacle inhibiting long-term graft survival in the pig-to-non-human primate transplant model. The present study was undertaken to determine whether repetitive injection of low dose Yunnan-cobra venom factor (Y-CVF), a potent complement inhibitor derived from the venom of Naja kaouthia can completely abrogate hemolytic complement activity and subsequently improve the results in a pig-to-rhesus monkey heterotopic heart transplant model. Nine adult rhesus monkeys received a heterotopic heart transplant from wild-type pigs and the recipients were allocated into two groups: group 1 (n = 4) received repetitive injection of low dose Y-CVF until the end of the study and group 2 (n = 5) did not receive Y-CVF. All recipients were treated with cyclosporine A (CsA), cyclophosphamide (CyP) and steroids. Repetitive Y-CVF treatment led to very dramatic fall in CH50 and serum C3 levels (CH50 < 3 units/C3 remained undetectable throughout the experiment) and successfully prevented hyperacute rejection (HAR), while three of five animals in group 2 underwent HAR. However, the continuous suppression of circulating complement did not prevent AVR and the grafts in group 1 survived from 8 to 13 days. Despite undetectable C3 in circulating blood, C3 deposition was present in these grafts. The venular thrombosis was the predominant histopathologic feature of AVR. We conclude that repetitive injection of low dose Y-CVF can be used to continuously suppress circulating complement in a very potent manner and successfully prevent HAR. However, this therapy did not inhibit complement deposition in the graft and failed to prevent AVR. These data suggest that using alternative pig donors [i.e. human decay accelerating factor (hDAF)-transgenic] in combination with the systemic use of complement inhibitors may be necessary to further control complement activation and improve survival in pig-to-non-human primate xenotransplant model.

Animals↗

PubMed2003

A new protein structure of P-II class snake venom metalloproteinases: it comprises metalloproteinase and disintegrin domains.

A new metalloproteinase-disintegrin, named Jerdonitin, was purified from Trimeresurus jerdonii venom with a molecular weight of 36 kDa on SDS-PAGE. It dose-dependently inhibited ADP-induced human platelet aggregation with IC(50) of 120nM. cDNA cloning and sequencing revealed that Jerdonitin belonged to the class II of snake venom metalloproteinases (SVMPs) (P-II class). Different from other P-II class SVMPs, metalloproteinase and disintegrin domains of its natural protein were not separated, confirmed by internal peptide sequencing. Compared to other P-II class SVMPs, Jerdonitin has two additional cysteines (Cys219 and Cys238) located in the spacer domain and disintegrin domain, respectively. They probably form a disulfide bond and therefore the metalloproteinase and disintegrin domains cannot be separated by posttranslationally processing. In summary, comparison of the amino acid sequences of Jerdonitin with those of other P-II class SVMPs by sequence alignment and phylogenetic analysis, in conjunction with natural protein structure data, suggested that it was a new type of P-II class SVMPs.

Amino Acid Sequence↗

PubMed2003

Prolonged cardiac xenograft survival in guinea pig-to-rat model by a highly active cobra venom factor.

A highly active cobra venom factor (CVF) was isolated from the venom of Naja kaouthia by sequential column chromatography. It displays strong anticomplementary activity, and has 1515 U of anticomplementary activity per mg protein. A single dose of 0.1 mg/kg CVF given i.v. to rats completely abrogated complement activity for nearly 5 days. Given 0.02 mg/kg of CVF, the complement activity of rats was reduced by more than 96.5% in 6 h. In guinea pig-to-rat heart transplant model, rats treated with a single dose of 0.05 mg/kg CVF had significantly prolonged xenograft survival (56.12+/-6.27 h in CVF-treated rats vs. 0.19+/-0.07 h in control rats, P<0.001).

Animals↗

PubMed2003

Purification, characterization and biological activity of an L-amino acid oxidase from Trimeresurus mucrosquamatus venom.

An L-amino acid oxidase (TM-LAO) from the venom of Hunan Trimeresurus mucrosquamatus was purified to homogeneity by three steps including DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration and Resource Q ion-exchange chromatography. TM-LAO is composed of two identical subunits with a molecular weight of 55 kD by SDS-polyacrylamide gel electrophoresis. The molecular weight was different with that of LAO purified from the same species distributed in Taiwan that was 70 kD. The 24 N-terminal amino acid sequence of TM-LAO is ADNKNPLEECFRETNYEEFLEIAR, which shares high similarity with other Viperid snake venom LAOs and has moderate similarity with Elapid snake venom LAOs. Further studies found that TM-LAO inhibited the growth of E. coli, S. aurues and B. dysenteriae. TM-LAO also showed cytotoxicity and platelet aggregation activity. All the biological activities were eliminated by catalase, a H(2)O(2) scavenger. It was shown that these biological effects were possibly due to the formation of H(2)O(2) produced by TM-LAO.

Amino Acid Oxidoreductases↗

PubMed2003

Characterization of a new bradykinin-potentiating peptide (TmF) from Trimeresurus mucrosquamatus.

A novel bradykinin-potentiating peptide (BPP), designated as TmF, has been purified to homogeneity from the venom of Trimeresurus mucrosquamatus by 70% cold methanol extraction, Sephadex G-15 gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). The amino acid sequence of TmF was determined to be pGlu-Gly-Arg-Pro-Leu-Gly-Pro-Pro-Ile-Pro-Pro (pGlu denotes pyroglutamic acid), which shared high homology with other BPPs. The molecular mass of TmF was 1.1107 kD as determinated by electrospray ionization-mass spectrometry (ESI-MS), which was in accordance with the calculated value of 1.1106 kD. The potentiating unit of TmF to bradykinin-induced (BK-induced) contraction on the guinea-pig ileum in vitro was (1.13 +/-0.3) unit (mg/L), and TmF (5.0 x10(-4) mg/kg) increased the pressure-lowering-effect of bradykinin (5.0 x10(-5 )mg/kg) with approximate descent value of (14 +/-2) mmHg. In addition, TmF inhibited the conversion of angiotensin I to angiotensin II, 2 x10(-3) mg of TmF caused 50% inhibition (IC(50)) of angiotensin- converting enzyme (ACE) hydrolyzing activity to bradykinin.

Amino Acid Sequence↗

PubMed2003

Alpha-neurotoxins of Naja atra and Naja kaouthia snakes in different regions.

Recent studies have shown that there are geographic variation of alpha-neurotoxins in Naja kaouthia, but the cause is not clear yet. In this work, venoms were collected from adult Naja atra in Zhejiang Province and Naja kaouthia in Yunnan Province, well identified by morphological characters and cytochrome b gene analysis in summer season to avoid age and seasonal variation in the venom composition. Then alpha-neurotoxins were purified and cloned from these two kinds of snakes. Three alpha-neurotoxins from Naja kaouthia (Yunnan) and two from Naja atra (Zhejiang) were identified. Together with previously reported alpha-neurotoxins in Naja kaouthia (Thailand) and Naja atra (Taiwan Province), it was found that the alpha-neurotoxins of Naja kaouthia in Yunnan Province were similar to those of Naja atra in Zhejiang and Taiwan Provinces, but different from those of Naja kaouthia in Thailand. This result can hardly be explained by population phylogeny or geographic distance. It might be due to the different climate, habitat and prey in Thailand in comparison with those in Yunnan, Zhejiang and Taiwan Provinces.

Amino Acid Sequence↗

PubMed2003

Jerdonase, a novel serine protease with kinin-releasing and fibrinogenolytic activity from Trimeresurus jerdonii venom.

A novel kinin-releasing and fibrin(ogen)olytic enzyme termed jerdonase was purified to homogeneity from the venom of Trimeresurus jerdonii by DEAE Sephadex A-50 anion exchange, Sephadex G-100 (superfine) gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). Jerdonase migrated as a single band with an approximate molecular weight of 55 kD under the reduced conditions and 53 kD under the non-reduced conditions. The enzyme was a glycoprotein containing 35.8% neutral carbohydrate. The N-terminal amino acid sequence of jerdonase was determined to be IIGGDECNINEHPFLVALYDA, which showed high sequence identity to other snake venom serine proteases. Jerdonase catalyzed the hydrolysis of BAEE, S-2238 and S-2302, which was inhibited by phenylmethylsulfonyl fluoride (PMSF), but not affected by ethylenediaminetetraacetic acid (EDTA). Jerdonase preferentially cleaved the A alpha-chain of human fibrinogen with lower activity towards B beta-chain. Moreover, the enzyme hydrolyzed bovine low-molecular-mass kininogen and releasing bradykinin. In conclusion, all results indicated that jerdonase was a multifunctional venom serine protease.

Amino Acid Sequence↗