莽山烙铁头蛇人工繁殖成功

1994～ 2 0 0 3年间对莽山烙铁头蛇进行人工孵化繁殖 ,总计孵出幼蛇 89条。从 2 0 0 0年起 ,孵化率最高达 79%。     

烙铁头蛇属一新种—莽山烙铁头蛇

本文描述在湖南宜章莽山发现的蝰科烙铁头蛇属的一新种。此蛇采到不久,中央电视台于1989年11月曾加以报道。     

莽山烙铁头蛇人工繁殖成功(图版Ⅹ)

1994～2003年间对莽山烙铁头蛇进行人工孵化繁殖,总计孵出幼蛇89条。从2000年起,孵化率最高达79％。     

莽山烙铁头蛇的濒危现状及保护对策

莽山烙铁头蛇地方名白尾蛇,是中国特有种,只在湘粤交界的南岭山脉骑田岭支脉的莽山地区一带有分布。由于分布狭窄,数量稀少,是一个极为脆弱的种群。在各种因素的干扰和破坏下,其数量正迅速减少,有濒临灭绝的危险。目前境况十分令人担扰,如果不迅速采取有效的保护措施,就有可能在较短的时间内绝灭。     

莽山烙铁头蛇Ermiamangshanensis(Zhao,1990)专项调查结果

1998－2000年,在莽山地区对莽山烙铁头蛇的分布、种群数量等进行了专项调查。调查结果表明莽山烙铁头蛇确实是一种数量稀少、分布范围狭窄的蛇类,已经处于极度濒危程度,建议尽快将其列为国家一级重点保护动物。     

莽山烙铁头蛇专项Ermia mangshanensis(Zhao,1990)调查结果

1998～ 2 0 0 0年 ,在莽山地区对莽山烙铁头蛇的分布、种群数量等进行了专项调查。调查结果表明莽山烙铁头蛇确实是一种数量稀少、分布范围狭窄的蛇类 ,已经处于极度濒危程度 ,建议尽快将其列为国家一级重点保护动物。     

莽山烙铁头蛇的资源调查报告

莽山烙铁头蛇 ( Ermia mangshanensis)是中国特有蛇种 ,于 1 989年在湖南莽山国家级自然保护区首次发现 ,1 990年被学术界正式命名 [1]。我们于 1 990～ 1 997年对莽山烙铁头蛇的分布及种群数量进行了初步的前期调查 ,于 1 998～ 2 0 0 0年对其进行了专项调查[2 ] ,现将调查结果报告如下。1　调查时间和方法1 .1　调查时间　　调查时间是 1 990～ 2 0 0 0年 ,其中前期调查是1 990～ 1 997年 ,专项调查是 1 998～ 2 0 0 0年。1 .2　调查方法　　前期调查以走访调查为主 ,实地调查为辅 ;专项调查以野外实地调查为主 ,兼顾走访调查。　　 ( 1 …     

莽山烙铁头蛇的养殖现状及存在问题

莽山烙铁头蛇分布范围狭窄,种群数量稀少,只在中国湘粤边界一带有分布,是中国特有的濒危蛇种。1994年列入《中国生物多样性保护行动计划》一级优先保护动物;1998年列入《中国濒危动物红皮书》极危等级的生物种;多次列入IUCN红色保护名录。开展对莽山烙铁头蛇的人工繁殖研究,扩大其种群数量,是保护好这一珍稀物种的唯一和关键的措施。我们从1994年起就开展了对莽山烙铁头蛇的人工繁殖和养殖研究,并成功繁殖100条莽山烙铁头幼蛇。在养殖莽山烙铁头蛇的过程中,我们发现有食物链狭窄、繁殖率低下、种群内亲杀、疾病与寄生虫等几种因素,严重影响莽山烙铁头蛇的成活率,这些因素的存在严重威胁着这个种群的生存。     

莽山烙铁头蛇的濒危现状与保护对策

目的探讨我国特有的珍稀蛇种──莽山烙铁头蛇的濒危现状与保护对策。方法基于文献资料,结合作者近年来的调查工作,对莽山烙铁头蛇的分布区、种群数量、濒危现状与威胁因素进行分析和探讨。结果近10年来,莽山烙铁头蛇的种群数量没有明显增加,基本保持稳定,但是仍遭受猎捕、贸易及栖息地破坏等因素的威胁。结论要保护好我国这一特有的珍稀蛇种,需要尽快将莽山烙铁头蛇列入国家重点保护野生动物名单,列入《濒危野生动植物种国际贸易公约》,加强法律保护力度及国际贸易管控,积极开展生态生物学研究,实施人工繁殖和放归野外计划,以促进野生种群复壮。     

心外按压复苏术救活莽山烙铁头蛇1例

蛇意外窒息,出现心跳呼吸停止后,为其行心外按压复苏术成功救活的,目前国内外尚未见报道。我们于2006年4月11日采用心外按压复苏术成功救活1例心跳呼吸停止15 min的莽山烙铁头蛇,现报告如下。     

Using Head Patch Pattern as a Reliable Biometric Character for Noninvasive Individual Recognition of an Endangered Pitviper Protobothrops mangshanensis

Mangshan pitviper, Protobothrops mangshanensis(formerly Zhaoermia mangshanensis) is endemic to China. Unfortunately, due to the decreasing size of its wild populations, this snake has been listed as critically endangered. Research carried out on the Mangshan pitviper's population ecology and captive reproduction has revealed that the unique head patch patterns of different individuals may potentially be used as a noninvasive recognition biometric character. We collected head patch pattern images of 40 individuals of P. mangshanensis between 1994 and 2011. By comparing each pitviper's head patch pattern, we found that the head patch pattern of individual snakes was different and unique. Additionally, we observed and recorded the head patch pattern characters of four adults and five juveniles before and after ecdysis. Our findings confirmed that head patch patterns of Mangshan pitvipers are unique and stable, remaining unchanged after ecdysis. Thus, individuals can be quickly identified by examining the head patch pattern within a specific recognition area on the head. This method may be useful for noninvasive individual recognition in many other species that display color patch pattern variations, especially in studies of endangered species where the use of invasive marking techniques is undesirable.     

Immunological properties of notexin, a potent presynaptic and myotoxic component from venom of the Australian tiger snake Notechis scutatus scutatus

Neutralizing antibodies were raised in mice against notexin, the most toxic phospholipase A₂ (PLA₂) from Notechis scutatus scutatus venom, without the necessity of detoxifying the toxin prior to immunization. Using a sensitive radioimmunoassay we demonstrated that anti-notexin antibodies recognized (i) the parent antigen, (ii) closely related isoforms of notexin and (iii) venoms from Notechis genus snakes. In contrast, they failed to recognize other purified PLA₂ or PLA₂-containing venoms from other origins. Substitutions or chemical modifications occurring in the C-terminal part of the polypeptide chain of notexin altered the binding affinity for antibodies, implying that this region constitutes an antigenic domain of notexin.     

Proteomic analyses of transgenic LQT1 and LQT2 rabbit hearts elucidate an increase in expression and activity of energy producing enzymes

Various biochemical and genomic mechanisms are considered to be a hallmark of metabolic remodeling in the stressed heart, including the hypertrophied and failing heart. In this study, we used quantitative proteomic 2-D Fluorescence Difference In-Gel Electrophoresis (2-D DIGE) in conjunction with mass spectrometry to demonstrate differential protein expression in the hearts of transgenic rabbit models of Long QT Syndrome 1 (LQT1) and Long QT Syndrome 2 (LQT2) as compared to littermate controls (LMC). The results of our proteomic analysis revealed upregulation of key metabolic enzymes involved in all pathways associated with ATP generation, including creatine kinase in both LQT1 and LQT2 rabbit hearts. Additionally, the expression of lamin-A protein was increased in both LQT1 and LQT2 rabbit hearts as was the expression of mitochondrial aldehyde dehydrogenase and desmoplakin in LQT1 and LQT 2 rabbit hearts, respectively. Results of the proteomic analysis also demonstrated down regulation in the expression of protein disulfide-isomerase A3 precuorsor and dynamin-like 120 kDa protein (mitochondrial) in LQT1, and of alpha-actinin 2 in LQT2 rabbit hearts. Up regulation of the expression of the enzymes associated with ATP generation was substantiated by the results of selective enzyme assays in LQT1 and LQT2 hearts, as compared to LMC, which revealed increases in the activities of glycogen phosphorylase (+50%, +65%, respectively), lactate dehydrogenase (+25%, +25%) pyruvate dehydrogenase (+31%, +22%), and succinate dehydrogenase (+32%, +60%). The activity of cytochrome c-oxidase, a marker for the mitochondrial function was also found to be significantly elevated (+80%) in LQT1 rabbit hearts as compared with LMC. Western blot analysis in LQT1 and LQT2 hearts compared to LMC revealed an increase in the expression of very-long chain-specific acyl-CoA dehydrogenase (+35%, +33%), a rate-limiting enzymes in β-oxidation of fatty acids. Collectively, our results demonstrate similar increases in the expression and activities of key ATP-generating enzymes in LQT1 and LQT2 rabbit hearts, suggesting an increased demand, and in turn, increased energy supply across the entire metabolic pathway by virtue of the upregulation of enzymes involved in energy generation.     

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.     

A novel phospholipase A2 from Agkistrodon blomhoffii ussurensis venom: Purification,proteomic, functional and structural characterizations

A phospholipase A₂ was isolated from the snake venom of Chinese Agkistrodon blomhoffii Ussurensis by column chromatography using DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration chromatography and Mono Q ion-exchange chromatography, and designated as Akbu-PLA₂. It showed an average molecular mass of 13,980 ± 3 amu determined by MALDI TOF mass spectrometry. Protein identification results from HPLC-nESI-MS/MS analysis indicated that the Akbu-PLA₂ was a new snake venom acidic PLA₂. Seven peptides were sequenced from Akbu-PLA₂ by HPLC-nESI-MS/MS analysis. Sequencing alignment indicated that Akbu-PLA₂ shared homolog peptides of phospholipases A₂ from the venoms of Gloydius ussurensis, Gloydius halys, Gloydius halys (halys viper), Deinagkistrodon acutus and Agkistrodon halys Pallas. Akbu-PLA₂ has an optimum hydrolytic activity temperature of ∼45 °C. The intrinsic fluorescences of Tyr and Trp residues of Akbu-PLA₂ showed emission wavelengths red-shifted by 13.6 and 1.6 nm from those of free Tyr and Trp, respectively. Akbu-PLA₂ was shown to contain one Ca²⁺ per monomer by ICP-AES measurement. The Ca²⁺ ion was found to be critical for both the hydrolytic activity and the structure of Akbu-PLA₂. Ca²⁺ increased the emission fluorescence intensity and the hydrophobicity of the environment of Akbu-PLA₂. The hydrolytic activity of Akbu-PLA₂ was accelerated due to the addition of Ca²⁺ ion by enhancing the substrate binding. However, a protein component with the molecular weight two-fold relative to that of Akbu-PLA₂ was found to be difficult to eliminate for the purification of Akbu-PLA₂. HPLC-nESI-MS/MS detected the same peptides from it as from Abku-PLA₂, which indicated that it should be a homodimer of Akbu-PLA₂. A proteomic approach, 2D SDS-PAGE coupled to HPLC-nESI-MS/MS, supported the co-existence of the Akbu-PLA₂ monomer and dimer in the crude snake venom. Results from the combination of phosphoprotein and glycoprotein specific stains combined with the HPLC-nESI-MS/MS method indicated that both the Akbu-PLA₂ monomer and dimer were both phosphorylated and glycosylated. The addition of exogenous Ca²⁺ ion was found to be able to promote the dimer formation of Akbu-PLA₂. We conclude that a novel PLA₂ was successfully obtained. The systemically biochemical, proteomic, structural and functional characterization results from Akbu-PLA₂ reveal new threads and provide valuable inputs for the study of snake venom phospholipases A₂.     

A New Species of the Genus Protobothrops(Squamata: Viperidae) from Southern Tibet,China and Sikkim,India

A new species of the genus Protobothrops Hoge Romano-Hoge, 1983, was described from Jilong County, southern Tibet, China, and Chungthang, northern Sikkim, India. It differs from congeners by the following characters: 1) relatively large body size(total length up to 1510 mm); 2) dorsal scale rows 25–25–19; 3) except for the smooth outermost row, dorsal scales are weakly keeled; 4) relatively high number of ventral(198–216) and subcaudal(65–76 pairs) scales; 5) 7–8 supralabials; 6) 11 to 13 infralabials; 7) dorsal head uniform dark brown, laterally a reddish-brown obscure postocular streak; 8) dorsum of trunk and tail olive, with distinct black edged red brown transverse bands across the body and tail; and 9) eye from bright brown and reddish brown to mildly brown. The new species was also observed from the Haa Valley in western Bhutan.     

Effect of phospholipase A on actions of cobra venom cardiotoxins on erythrocytes and skeletal muscle

The actions of two phospholipase-free cardiotoxins from the venom of the cobra Naja naja siamensis were compared to phospholipase-contaminated cardiotoxins in terms of their ability to lyse human erythrocytes and to depolarize and contract skeletal muscle. The presence of 3–5% (w/w) phospholipase caused a 20–30-fold increase in the haemolytic activity of the two cardiotoxins, the pure cardiotoxins being virtually without haemolytic activity at 10^?7-10^?6 M. Phospholipase contamination did not enhance the ability of the cardiotoxins to cause contracture of chick biventer cervicis muscles and it caused less than a 2-fold increase in the depolarizing activity of the cardiotoxins on cultured skeletal muscle. Phospholipase-free cardiotoxins were about 10–20-times more active on cultured skeletal muscle fibres than on erythrocytes. These results support the hypothesis that some cardiotoxins have more affinity for the membranes of excitable cells than for those of other cells such as erythrocytes.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E₁ or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. $\text{p-}\text{Bromophenacyl}$ bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: $\text{arachidonic acid}\text{>}\text{collagen}\text{>}\text{thrombin}\text{>}\text{ionophore A}\text{23187}$. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca²⁺ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca²⁺ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Mutagenesis analyses explore residues responsible for the neurotoxic and anticoagulant activities of Trimucrotoxin, a pit-viper venom Asn6-phospholipase A2

Trimucrotoxin (TmCT) is an Asn⁶-containing phospholipase A₂ (PLA₂) from Protobothrops mucrosquamatus (pit-viper) venom. In an attempt to characterize the amino acid residues responsible for the neurotoxic and anticoagulant activities of TmCT, the recombinant fusion proteins of TmCT wild type and mutants were expressed in Escherichia coli. Correct refolding and processing of 37 TmCT mutants were confirmed by their HPLC retention times, circular dichroism spectra, and masses obtained from ESI-MS spectrometry. Each mutant was assayed by pH-stat titration using zwitterionic as well as anionic micelle substrates, and the neurotoxicity was evaluated by using the contractile responses of chick biventer cervicis muscles. The results demonstrated that the residues Asn¹, Asn⁶, Lys⁷, Ile¹¹, Met¹², Gly⁵³, Thr⁷⁹, His¹⁰⁸ and Met¹¹⁸ are important to TmCT neurotoxicity. Through various tests, we also confirmed that enzymatic activity, as opposed to binding to Factor Xa, was a necessary part of TmCT’s anticoagulant effect. In addition, pulldown assays of the WT and selected mutants revealed that TmCT’s in vitro binding to crotoxin acidic subunit may involve a broad surface area. We conclude that the hot spot mutations at specific positions 53, 79, 108, and 118 during the pit-viper Asn⁶-PLA₂ evolution regulate their neurotoxicities, and that many of the neurotoxic site residues and the anticoagulant mechanism of TmCT are different from those of ammodytoxin A (a true-viper venom neurotoxic PLA₂).     

Phosphatidic acid as the biosynthetic precursor of the endocannabinoid 2-arachidonoylglycerol in intact mouse neuroblastoma cells stimulated with ionomycin

In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2-arachidonoylglycerol (2-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2-AG. [3H]2-AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg2+/Ca2(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]2-AG formation. These data indicate that DAG precursors for 2-AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]2-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.